DharmaconTM. RNAi & Gene Expression. Product Guide 2014

Transcription

1 DharmaconTM RNAi & Gene Expression Product Guide 214

2 Every discovery is a stepping stone toward clarity, firm conclusions, and ultimately an amazing breakthrough. With so much riding on every experiment, why take a chance? Set yourself up for success with Dharmacon. With our RNAi and gene expression products, you can gain a full picture of what your gene is doing and what that means for you. But there s more to Dharmacon than just scientific tools. You re also gaining a lab partner. Someone who can provide recommendations for experimental design, troubleshoot, and celebrate your findings. Knock out uncertainty with Dharmacon. High confidence discoveries are now yours.

3 Solutions for RNAi and Gene Expression Dharmacon RNAi and gene expression products encompass the most comprehensive portfolio of tools for gene knockdown and over-expression. Our innovative product portfolio contains industry-leading, shrna, microrna, cdna and ORF reagents as well as a complete suite of products for delivery, qpcr reagents, controls and confirmation of results. Dharmacon continues to be the industry leader in the field of RNA chemistry and RNAi biology, and partners with the international RNAi screening community through participation in the RNAi Global Initiative. More information about RNAi Global on p. 75 or visit rnaiglobal.org From single gene analysis to genome-scale screens, find your RNAi and Gene Expression solutions at gelifesciences.com/dharmacon Patented dual-strand modifications in ON-TARGETplus provide unrivaled specificity and potency; patented Accell is the only reagent that can be delivered to difficult-to-transfect cells without a transfection reagent shrna microrna-adapted hairpin designs plus constitutive and inducible options allow for simple, specific and potent silencing in many cell lines microrna Up-regulate or suppress endogenous mature microrna function with rationally designed synthetic and expressed miridian microrna Mimics and Hairpin Inhibitors cdnas and ORFs Choose from large collections of pre-made cdna and ORF clones for gene expression and RNAi rescue experiments 2 RNAi Technologies RNAi Technologies 3

4 Table of Contents Solutions for RNAi and Gene Expression 2 Section I - RNAi Solutions 6 Introduction to RNAi Biology 8 tools 1 shrna tools 11 microrna tools 12 RNAi application selection guide 14 Technologies 16 selection guide 17 Strategies for premium specificity and guaranteed silencing 18 ON-TARGETplus 2 Accell 22 sigenome 24 ordering guide 25 Lincode for lncrna 26 Cherry-pick RNAi Libraries 27 shrna Technologies 28 shrna selection guide 29 Less toxicity, more specificity with microrna-adapted shrna 3 SMARTchoice Lentiviral shrna 32 SMARTchoice Inducible Lentiviral shrna 34 GIPZ Lentiviral shrna 35 TRIPZ Inducible Lentiviral shrna 36 TRC Lentiviral shrna 37 GIPZ and TRIPZ shrna Starter Kits 38 High-titer lentiviral particle production 39 microrna Modulation Technologies 4 microrna research tools selection guide 41 SMARTchoice shmimic Lentiviral microrna 42 miridian microrna Mimics 44 miridian microrna Hairpin Inhibitors 45 Custom Oligonucleotide Synthesis 46 Custom synthesis selection guide 47 Custom synthesis 48 sidesign Center 49 reagents for in vivo applications 5 Custom microrna modulation tools 51 Custom RNA synthesis selection guide 52 Custom single-stranded RNA synthesis 53 RNAi Controls 54 RNAi Controls selection guide 55 Controls 56 ON-TARGETplus Controls 56 Accell Controls 56 sigenome Controls 56 sistable Controls 56 siglo Fluorescent Controls 56 Additional Positive Controls 56 Lincode controls 57 Find the right products using the following selection guides RNAi application selection guide p. 14 selection guide p. 17 shrna selection guide p. 29 microrna selection guide p. 41 Custom synthesis selection guide p. 47 RNAi Controls selection guide p. 55 Delivery method selection guide p. 59 RNAi Libraries selection guide p. 67 cdna & ORF selection guide p. 81 Model organism selection guide p RNAi Controls (continued) shrna Controls 57 SMARTchoice Lentiviral shrna Controls 57 SMARTchoice Inducible Lentiviral shrna Controls 57 GIPZ Lentiviral shrna Controls 57 TRIPZ Inducible Lentiviral shrna Controls 57 TRC Lentiviral shrna Controls 57 microrna Controls shmimic Lentiviral microrna Controls 57 miridian microrna Mimic Controls 57 miridian microrna Hairpin Inhibitor Controls 57 Delivery Solutions for RNAi 58 Delivery method selection guide 59 DharmaFECT Transfection Reagents 6 TurboFect Transfection Reagent 62 Trans-Lentiviral shrna Packaging Kits 63 Ancillary Reagents 64 Stability testing of DharmaFECT Transfection Reagents 65 RNAi Screening 66 RNAi Libraries selection guide 67 Pre-defined Gene Family and Pathway Libraries 68 Reverse Transfection Format (RTF) Libraries 7 Duet Libraries 72 Decode Pooled Lentiviral shrna Libraries 73 miridian microrna Mimic and Hairpin Inhibitor Libraries 74 RNAi screening across the workflow 75 Section II - Gene Expression 76 Introduction to Gene Expression 78 Gene over-expression as a tool for gene analysis and protein science 78 The importance of full insert sequencing 79 Save time and money with pre-made cdna and ORF clones 79 Pre-made cdnas and ORFs 8 cdna and ORF selection guide 81 Mammalian Gene Collection 83 CCSB Human ORFeome Collection 83 Human ORFeome V ORFeome Collaboration Collection 85 CCSB-Broad Lentiviral Expression Library 86 Precision LentiORF Collection 87 Pre-defined Gene Family cdna & ORF Libraries 88 TurboFect Transfection Reagent 89 TurboFect in vivo Transfection Reagent 9 Trans-Lentiviral ORF Packaging Kit 91 Non-mammalian Resources 92 Model organism selection guide 94 Yeast Collections 95 Molecular Barcoded Yeast ORFs 96 E. coli Collections 97 C. elegans Collections 98 Xenopus Gene Collection 99 Zebrafish Gene Collections 99 Appendix 1 Frequently asked questions concerning, shrna, microrna, custom oligonucleotide synthesis, gene expression 11 Trademarks and license information 19 Glossary 11 Index by term 111 Index by product name 112 Key to Icons Human Mouse Rat Bacterial glycerol stock SMARTpool 4 Set of four 1 GFP RFP Individual; or microrna Gene families and pathways Arrayed library format High-titer viral particles Starter kits Green fluorescent marker Red fluorescent marker Additional information 4 RNAi Technologies RNAi Technologies 5

5 Section I RNAi Solutions Chapter 1: Introduction to RNAi Biology Chapter 2: Technologies Chapter 3: shrna Technologies Chapter 4: microrna Modulation Technologies Chapter 5: Custom Oligonucleotide Synthesis Chapter 6: RNAi Controls Chapter 7: Delivery Solutions for RNAi Chapter 8: RNAi Screening Libraries RNAi Technologies 7

6 RNAi Biology I.1 Introduction to RNAi Biology Harnessing the endogenous microrna pathway I.1 RNAi Biology RNA interference (RNAi) is a form of post-transcriptional gene silencing (PTGS) mediated by two types of small non-coding RNAs: small interfering RNAs (s) and micrornas. 1, 2 RNAi is observed in a broad range of eukaryotic species and is one manifestation of an expanding list of gene regulatory processes mediated by RNA. 3, 4 The original phenotypic observations of RNAi were made more than twenty years ago by researchers working in plant and fungal genetics. 5 Over the last decade, major strides have been made in understanding the mechanism and implications of RNAi, and in 26 Andrew Fire and Craig Mello won the Nobel Prize for their work towards this end. Working in C. elegans, they observed that injection of double-stranded RNA (dsrna) induced sequence-specific reduction of a target mrna. Moreover, they found that only a few molecules were required to reduce the population of target mrna, suggesting an extremely potent mechanism of action. 6 In nature, RNAi can be initiated by exogenous sources, such as long dsrna transcribed from invading viruses or by transcription of endogenously encoded micrornas. The predominant source of RNAi in mammalian somatic cells is the microrna pathway (Figure 1). Long primary micrornas (pri-microrna) are transcribed in the nucleus, where the Microprocessor (Drosha/ DGCR8) mediates processing of pri-microrna transcripts into precursor micrornas (pre-micrornas) that are then exported to the cytoplasm by Exportin-5. 7 Dicer cleaves these pre-micrornas into small double stranded RNAs of approximately 23 base pairs (bp). 8 These small RNAs are loaded into the RNA-Induced Silencing Complex (RISC), whereupon a RISC-associated, ATP-dependent helicase activity unwinds the duplex, thus enabling the active strand to guide target mrna recognition. Upon identification of the mrna target, the Argonaute protein in the RISC will either direct catalytic endonucleolytic cleavage of an mrna target (in the rare case of full base pairing complementarity), or direct the mrna for degradation and/ or translational repression by binding a short, six to eight base region located at the 5' terminus of the microrna referred to as the seed region. 9 The ultimate result of both types of microrna regulation is gene silencing and down-regulation of protein expression. Nucleus pri-microrna microrna Pathway Microprocessor (Drosha/DGCR8) pre-microrna Derived Tools microrna-adapted shrna microrna over-expression constructs Stem-loop shrna Exportin-5 Cytoplasm Dicer complex Harnessing RNAi as a tool RNAi revolutionized the study of biology, providing scientists with tools to silence individual genes in a sequence-specific manner. In 21, two groups independently demonstrated that short, synthetic RNA duplexes, termed small interfering RNAs (s), could serve as functional silencing intermediates in the endogenous mammalian microrna pathway. When introduced into cultured cells, s were shown to elicit potent, sequencespecific inhibition of target mrna without induction of the interferon response, which would result in general repression of all protein synthesis if longer dsrna were delivered. 1,11 Shortly thereafter, several groups published evidence that RNAi could also be effectively achieved in mammalian cells by transfection of plasmids encoding short hairpin RNAs (shrnas), small RNA transcripts consisting of 19- to 29-bp stems bridged by four to nine nucleotide loops The development of viral vector-based expression of shrna constructs quickly followed and broadened RNAi experiments into systems that a) require extended duration of silencing, b) involve cells intractable to lipid-based delivery, or c) utilize animal models. microrna tools were also developed, in the form of mimics and inhibitors, to copy or interfere with native microrna activity, thus providing techniques to evaluate the endogenous function of these conserved non-coding transcripts. As tools for exogenously inducing RNAi were developed, researchers recognized the utility of RNAi as an elegant and valuable mechanism for systematic gene functional analyses. The result has been a substantial shift from traditional gene knockout, antisense or ribozyme technologies towards implementing RNAi as a standard technique for discovery biology and target validation. The complete sequence complementarity of - or shrna-mediated RNAi, or the partial complementarity of micrornas permits experimental strategies for the evaluation of discrete genes, splice variants or closely related family members. As such, these tools are now routinely used to perform individual gene functional analyses, as well as to dissect complex biological pathways or survey entire genomes in a high-throughput manner. What follows in this chapter is a brief description of the founding molecules, shrna and microrna from which a variety of rationally designed, synthetic, modified and/or expressed silencing intermediates have been derived. Chapters 2-8 describe reagents that are commercially available for effecting gene silencing, as well as ancillary products for the delivery of these tools and assessment of their activity. Section II completes the list of reagents available for gene modulation studies, including cdna and ORF over-expression. Mature microrna (1) mrna degradation/ translational repression RISC (2) Catalytic mrna cleavage Synthetic or microrna mimic microrna inhibitor Figure 1. The endogenous mammalian microrna pathway (orange molecules) starting with transcription of the pri-microrna in the nucleus and ending with two options for gene down-regulation: (1) seed-mediated mrna degradation or translational repression and (2) catalytic mrna cleavage mediated by full sequence complementarity. The blue molecules to the right of the image illustrate a variety of RNAi tools that have been developed and where they enter the endogenous RNAi mechanism. 8 RNAi Biology RNAi Biology 9

7 RNAi Biology I.1 tools shrna tools I.1 RNAi Biology Design and mechanism Design and mechanism Synthetic s are the most commonly used RNAi tools for inducing endogenous gene silencing. s consist of two RNA strands, an antisense (or guide) strand and a sense (or passenger) strand which form a duplex 19 to 25 bp in length with 3' dinucleotide overhangs (Figure 1 p. 9). Small double-stranded s are transfected into cells where the guide strand is loaded into RISC. This activated protein and nucleic acid complex can then elicit gene silencing by binding, through perfect complementarity, to an mrna target. Synthetic s are most commonly generated through solid-phase chemical synthesis methods (such as 2'-ACE chemistry patented by Dharmacon, Inc., p. 46) which provide highly pure, stable, and readily modified s. Function The success of RNAi experiments depends on the efficiency of gene knockdown. Early work on design established conventional guidelines for structural attributes that led to reasonably functional knockdown in specific cases. 1 The properties of reliably potent s were further refined by performing large- scale functional studies that defined thermodynamic and sequencebased rules for rational design. 15 These design algorithms greatly improved the frequency of identifying potent sequences. SMARTselection technology was the first comprehensive rational design strategy applied to commercial collections, resulting in the production of reliably potent ( further described in Chapter 2, pages 16-27). While research is continually striving to identify molecules with greater activity and specificity, s designed by SMARTselection strategies, such as sigenome and ON-TARGETplus reagents, remain the most effective reagents on the market ( p. 18). Specificity Although the sequence complementarity-based mechanism underlying RNAi allows for targetspecific gene knockdown, the same mechanism can result in s that induce undesired changes in expression profiles. Several strategies have been developed to mitigate these so-called off-target effects and ensure on-target activity. Chemical modifications to the have been used successfully to promote preferential loading of the intended antisense (guide) strand into the RISC complex 16, 17 and reduce sense (passenger) strand loading and 18, 19 activity. Furthermore, to reduce the risk of the guide strand seed region from causing off-target effects, design algorithms can incorporate filters that exclude seed sequences from known mammalian micrornas. 2 Chemical or thermodynamicbased modifications can also be applied to the seed region to discourage undesired interactions. 19, 21, 22 Finally, the strategy of pooling several independent s that target an individual gene has been shown to reduce the total number of non-specific gene targets and the frequency of off-target phenotypes while preserving potent target gene knockdown. 23 All of these strategies, when combined, work efficiently to reduce off-targeting and to achieve potent silencing for a successful RNAi experiment. Applications s are widely used to assess the individual contributions of genes to an assortment of cellular phenotypes including cytokinesis, 24 apoptosis, 25 insulin signaling 26, 27 and cell differentiation. 28 screens have been used to identify novel pathways 29 and have had significant impact in validating targets for a number of cellular processes and diseases including cancer, 3, 31 HIV infection 32 and hepatitis. 33 Finally, in vivo RNAi has been used for target validation studies in animal disease models and has the potential to be used for therapeutic purposes where disease-causing genes are selectively targeted and suppressed. 34 shrna sequences are generally encoded in a DNA vector that can be introduced into cells by plasmid transfection or viral transduction. shrna molecules can be divided into two main categories based on their designs: simple stem-loop and microrna-adapted shrna. A simple stem-loop shrna is often transcribed under the control of an RNA Polymerase III (Pol III) promoter. 35, 36 The 5-7 nucleotide transcript forms a stem-loop structure consisting of a 19 to 29 bp region of double stranded RNA (the stem) bridged by a region of predominantly single-stranded RNA (the loop) and a dinucleotide 3' overhang The simple stem-loop shrna is transcribed in the nucleus and enters the pathway similar to a pre-microrna ( p. 31). The longer (> 25 nucleotide) microrna-adapted shrna is a design that more closely resembles native pri-microrna molecules, consisting of an shrna stem structure which may include microrna-like mismatches, bridged by a loop and flanked by 5' and 3' endogenous microrna sequences. 4 The microrna-adapted shrna, like the simple stem-loop hairpin, is also transcribed in the nucleus but is thought to proceed earlier through the RNAi mechanism similar to an endogenous pri-microrna. shrna technologies are DNA-based, which provides flexibility in design of the vector. Most vector-based shrna systems contain a selectable marker to allow for the elimination of cells that have not been Function Optimal gene knockdown is a requirement for successful RNAi using shrna systems. Rational design of shrna sequences has largely been based on algorithms developed with. While some design rules apply to shrna, more refined shrna design algorithms will likely improve target gene silencing for shrnas in the future. 47 In order to predict functional shrna sequences, the SMARTvector shrna algorithm selects target sequences on the basis of numerous criteria, including position-dependent nucleotide preferences, secondary structure and thermodynamic stability profiles specific to the SMARTvector microrna-based scaffold. Additionally, the SMARTvector algorithm includes several criteria for increasing specificity. Specificity As with s, bioinformatic approaches can be applied to create target-specific shrnas while minimizing the potential for off-target effects. High concentrations of silencing intermediates are known to contribute to off-target events, but the level of the intermediates are difficult to control when shrnas are exogenously expressed. Several publications have documented evidence that, under specific conditions, high levels of simple stem-loop shrna expression may saturate the endogenous RNAi pathway, leading to unintended phenotypes. 48 Other studies have suggested that the use of a microrna-adapted shrna may reduce cellular toxicity for in vivo RNAi experiments 4, 49 due to these scaffolds being more efficiently processed by both Microprocessor 5, 51 and Dicer. successfully transfected or transduced, and maintenance of cells with sustained gene knockdown. The shrna expression cassettes can also be incorporated into viral vector systems, including retrovirus, adenoassociated virus, adenovirus and lentivirus, which permit incorporation into, and expression from, the host genome. These viral strategies permit for shrna delivery to cell lines that are refractory to transfection. Fluorescent markers (such as a Green or Red Fluorescent Protein [GFP or RFP]) can also be included for tracking cells expressing shrnas ( see available SMARTchoice, GIPZ and TRIPZ shrna on pages 32 and 36). The performance of shrnas is influenced by many factors including the efficiency of transduction or transfection, the promoter driving expression of the shrna and epigenetic modifications (which can lead to silencing of shrna expression). Furthermore, the influence that each of these factors have on vector performance can differ depending on the cell line or cell type used SMARTchoice shrna options are available to help researchers select optimal promoters for shrna expression and gene silencing ( p. 32). Finally, when these shrna expression cassettes are coupled with inducible promoters, as with SMARTchoice Inducible Lentiviral shrna or the TRIPZ vector system ( p. 34 and 36), researchers can design studies to temporally and spatially modulate gene expression Applications shrnas offer the possibility for prolonged gene silencing. Transduction of virally packaged shrnas allows access to cells, such as primary and neuronal cells, that are difficult to transfect by traditional cationic lipid-based strategies. Virally delivered shrnas have also been used to evaluate whole genomes using pools of silencing constructs, and are now widely used to perform highthroughput screens to identify genes required in a variety of processes such as cancer cell survival and proliferation, tumor suppressor pathway components, 55 modulators of the mammalian circadian clock, 56 suppressors of epithelial mesenchymal transition, 57 host 58, 59 mediators of HIV-1 replication, and regulators of cell migration. 6 The power of pooled RNAi screening has also been extended to screening for gene function in animal models 61, 62 to investigate in vivo biology. 1 RNAi Biology RNAi Biology 11

8 RNAi Biology I.1 microrna tools Design and mechanism Aside from the adoption of the RNAi pathway for - and shrnainduced gene silencing, interest remains in determining the function of endogenous micrornas. 63 micrornas can target hundreds of genes simultaneously, 64 inducing subtle but reproducible shifts in gene expression and regulation of a variety of biological processes. Synthetic or expressed microrna mimics and inhibitors can be valuable tools for modulating microrna expression, thus helping to reveal the role of micrornas in development, differentiation and disease ( p. 74). microrna Mimics Two types of microrna mimetics are used to conduct gain-offunction experiments: synthetic mimics and over-expression constructs. Synthetic microrna mimics are chemically synthesized, double-stranded RNA molecules that are intended to mimic the transient duplexed product of Dicer complex processing ( p. 9). One strand is designed to load as the mature microrna as annotated in mirbase ( Like naturally occurring micrornas, the two strands of a synthetic mimic separate, the single-stranded mature microrna is incorporated into RISC and thereby directs downregulation of transcript levels. Synthetic microrna mimics require delivery into cells via transfection ( p. 58). Over-expression constructs are plasmids that encode native microrna sequences to achieve exogenously introduced microrna Function In contrast to and shrna tools, whose designs are generally not restricted by limitations of sequence space within the target mrna, microrna tools are based on sequences explicitly defined in mirbase and are generally not altered in order to preserve native function. Instead, alternative strategies are applied for enhancing activity of mimics and inhibitors. Both synthetic and expressed microrna mimics function by increasing the abundance of mature microrna sequences in a cell. To improve mimics, chemical modifications and structural enhancement strategies are applied for improving mature (or guide) strand RISC loading. Similarly, the function of microrna inhibitors can be enhanced by modifying the inhibitor structure or using different chemical modifications to improve binding to matrure microrna Specificity The specificity of micrornas is based on their mature sequence and is achieved through complementarity of target mrnas with the seed region of the microrna that is only six nucleotides in length. Consequently, one microrna can target many different genes and the mammalian RNAi mechanism relies on this multiple targeting to regulate phenotypes. Since microrna mimics predominantly load and utilize the mature sequence, these tools mimic the specificity of the mature microrna strand. In addition, some micrornas are closely related in sequence (termed family members, and often have redundant function. 74 Similarly, there is potential for some cross-reactivity of inhibitors to several micrornas when sequences are closely related. 75 expression. Similar to shrna constructs, plasmid DNA can be directly transfected into the cells, or plasmid vectors can be engineered to encode microrna sequences in the context of a viral backbone. Thus, by introducing these molecular mimics into a cell type of interest, one can enhance or supplement endogenous microrna activity ( p ). microrna Inhibitors When studying the functional role of a microrna, one should seek to characterize microrna effects by both gain-of-function and lossof-function observations. The introduction of microrna inhibitors into a biological system results in a loss-of-function assay with a predicted decrease in endogenous microrna function. Two types of inhibitors are available: synthetic and expressed inhibitors. Synthetic inhibitors are generally a non-hydrolyzable, single-stranded reverse complement to the mature microrna. The mechanism of inhibition is likely mediated by irreversible binding of the inhibitor to mature microrna-loaded RISC, thus preventing interaction of the mature microrna to its endogenous targets ( p. 45). Expressed inhibitors (sometimes referred to as microrna sponges) are generally artificial mrna constructs with multiple microrna sites that compete with natural mrna target for binding of micrornas. 64, 7 A combination of these approaches and tools in loss-of-function assays increase the likelihood of observing the otherwise subtle phenotypes often associated with microrna inhibition. Applications microrna mimics and inhibitors are essential tools to perturb microrna function and assess the phenotypic consequences in gain- or loss-of-function assays. microrna mimics and inhibitors can be used in conjunction with microrna and gene expression profiling for identification of specific microrna-gene target relationships. In addition, several screens have been performed that demonstrate microrna involvement in biological systems such as cancer and viral infection. 68, 69 While both microrna mimics and inhibitors have potential for studies in vivo, microrna inhibitors in particular have shown promise in therapeutic 62, development. References 1. Zamore, P.D. and B. Haley (25) Ribo-gnome: The big world of small RNAs. Science 39(574): Carthew, R.W. and Sontheimer, E.J. (29) Origins and mechanisms of mirnas and s. Cell 136: Amaral, P.P. et al. (28) The eukaryotic genome as an RNA machine. Science 319(5871): Ketting, R.F. (211) The many faces of RNAi. 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(211) Therapeutic inhibition of mir-28a improves cardiac function and survival during heart failure. Circulation 124(14): Huntzinger, E. and Izaurralde, E. (211) Gene silencing by micrornas: contributions of translational repression and mrna decay. Nature Reviews Genetics 12(2): Friedman, R.C. et al. (29) Most mammalian mrnas are conserved targets of micrornas. Genome Research 19(1): Lim, L. et al. (25) Microarray analysis shows that some micrornas downregulate large numbers of target mrnas. Nature 433: Krutzfeldt, J. et al. (25) Silencing of micrornas in vivo with 'antagomirs'. Nature 438: Horwich, M.D. and Zamore, P.D. (28) Design and delivery of antisense oligonucleotides to block microrna function in cultured Drosophila and human cells. Nature Protocols 3: Wang, Y. et al. (28) Embryonic stem cell-specific micrornas regulate the G1-S transition and promote rapid proliferation. Nature Genetics 4(12): Santhakumar, D. et al. (21) Combined agonist-antagonist genome-wide functional screening identifies broadly active antiviral micrornas. PNAS 17(31): Thomas, M. et al. (21) Desperately seeking microrna targets. Nature Structural & Molecular Biology 17(1): Vermeulen, A. et al. (27) Double-stranded regions are essential design components of potent inhibitors of RISC function. RNA 13(5): Davis, S. et al. (26) Improved targeting of mirna with antisense oligonucleotides. Nucleic Acids Research 34: Ørom, U.A. et al. (26) LNA-modified oligonucleotides mediate specific inhibition of microrna function. Gene 372: Gregory, P.A. et al. (28) The mir-2 family and mir-25 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nature Cell Biology 1: Robertson, B. et al. (21) Specificity and functionality of microrna inhibitors. Silence. 1(1): Montgomery, R. and van Rooij, E. (211) Therapeutic advances in MicroRNA targeting. Journal of Cardiovascular Pharmacology. 57(1): Elmen, J. et al. (28) Antagonism of microrna-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mrnas in the liver. Nucleic Acids Research 36(4): Trang, P. et al. (211) Systemic delivery of tumor suppressor microrna mimics using a neutral lipid emulsion inhibits lung tumors in mice. Molecular Therapy 19(6): Petri, A. et al. (29) MicroRNA Silencing in primates: towards development of novel therapeutics. Cancer Research 69(2): I.1 RNAi Biology 12 RNAi Biology RNAi Biology 13

9 RNAi Biology I.1 RNAi application selection guide I.1 RNAi Biology Choose your RNAi application to find the product solution that best suits your experimental needs. Follow the colored box to find the chapter that details best-in-class, shrna and microrna reagents. Transient or short-term RNAi Stable or long-term RNAi In vivo RNAi Inducible RNAi Modulation of non-coding RNA Are your cells difficult-totransfect? lncrna microrna Accell : modified for delivery to difficult-totransfect cells Learn more about Accell on page 22 Yes Transduce shrna and select Learn more about shrna reagents on page 29 Transfect Learn more about reagents on page 17 No Learn more about shrna reagents on page 29 Transduce or transfect shrna Transduce shrna and select Learn more about shrna reagents on page 29 Transduce shrna hightiter lentiviral particles Learn more about shrna reagents on page 29 Deliver stabilized Learn more about stabilized on page 5 Transduce or transfect Inducible shrna Learn more about inducible shrna on pages 34 & 36 Transfect targeting lncrna Learn more about Lincode on page 26 Yes Transduce lentiviral microrna Learn more about SMARTchoice shmimic microrna on page 42 Are your cells difficult-totransfect? No Transfect microrna mimics or inhibitors Learn more about miridian microrna reagents on page 44 The use of antibiotic selection to purify a population of shrna-expressing cells based on the shrna-containing vector also containing an antibiotic resistance gene. 14 RNAi Biology RNAi Biology 15

10 Technologies I.2 I.2 Technologies Which Dharmacon is best for you? Technologies The most trusted and comprehensive offering of pre-designed, genome-wide products available; providing enhanced specificity and guaranteed* silencing. Target long noncoding RNA Target protein-coding genes ON-TARGETplus sigenome Accell Lincode Species Guaranteed silencing* Seed region analysis for specificity Dual-strand modification for improved specificity The only single reagent that provides guaranteed knockdown Achieve potent gene silencing with improved target specificity page 19 Recommended for primary and non dividing cells including neuronal and immunological cells Patent-pending modification for delivery without transfection reagents or electroporation Stabilized for in vivo RNAi Extended duration silencing via repeated dosing Whole genome library availability pages Druggable genome library availability Deliver into difficult-to-transfect cell types without a transfection reagent A patented dual-strand modification to address the primary source of off targets pages Cherry-pick library availability** 2-21 Learn more on page number 24 Follow the icons to your product solution SMARTpool: A mixture of four s provided as a single reagent. sigenome and ON-TARGETplus SMARTpool reagents are guaranteed* to silence by 75% or better. page 2-21 Table of Contents selection guide Strategies for premium specificity and guaranteed silencing ON-TARGETplus... 2 Accell sigenome Formats for all 4 Set of Four: Four unique s targeting a single gene provided as individual reagents. Three of four sigenome and ON-TARGETplus s are guaranteed* to silence by 75% or better. Species: Individual : Human Mouse 1 Eight guaranteed reagents per gene Select one, two, three or four individual s per gene at a variety of quantities. Gene families and pathways: Pre-defined libraries arranged by gene family and/or function. Custom libraries: Available in 96-well or 384-well microtiter plates.** Rat ordering guide Lincode for lncrna Cherry-pick RNAi Libraries...27 Technologies 16 * s igenome and ON-TARGETplus reagents (SMARTpool and at least three of four individual s) are guaranteed to silence target gene expression by at least 75% at the mrna level when used under optimal delivery conditions (confirmed using validated positive and negative control s). Silencing should be monitored at the mrna level hours after transfection using 1 nm. ** M inimum order requirement of 2 wells per order. Learn more about how to order on p. 66. Technologies 17

11 Technologies I.2 Strategies for premium specificity & guaranteed silencing products are the result of scientific innovation in design and modification strategies to optimize potency, specificity and delivery. SMARTselection design Our scientists were the first to establish design rules for high potency silencing 1 and continue to drive innovation in this area 2,3. By providing genome-wide s designed with the SMARTselection algorithm, researchers can focus on successful gene silencing experiments instead of optimizing designs for their genes of interest. SMARTpool technology: improves specificity AND potency Pooling of s better mimics the natural RNAi pathway, and when combined with SMARTselection design, provides advantages for both potency and specificity. Targeting four mrna regions at once improves the likelihood of effective silencing and reduces false negatives due to inaccessible binding sites Lower relative concentration of each reduces sequence-specific off-targeting sigenome, ON-TARGETplus and Accell products are all available as SMARTpool reagents or up to 4 individual s Microarray analysis demonstrates SMARTpool potency and reduced off targets. Target gene silencing is maintained while off-target signatures are reduced with a SMARTpool reagent when compared to its four constituent s. Total RNA for microarray extracted from HeLa cells 24 hours posttransfection. Green indicates mrna reduced by two-fold or more (Agilent 22K Platform). Reduce off-targets caused by the seed region Target Target mrna AAAA SMARTpool I.2 Technologies The principles of SMARTselection design Dharmacon products provide the most comprehensive bioinformatic strategies to mitigate off-targeting. Build list of candidate 19 mer sequences that target the appropriate region(s) of the target gene Filter to remove candidates with known functionality or specificity issues (G/C content, SNP, toxic or microrna-like seed motifs, etc.) Score remaining candidates with proprietary algorithm (high score = high silencing potential) Crop list of candidates to eliminate low scores and increase efficiency of downstream analyses BLAST both strands to identify potential unintended targets Pick the four best with a proprietary set of criteria for seed region frequency, Score, BLAST results and targeting region The primary source of -mediated off targets is the seed region 2 (nucleotides 2-7) which uses the microrna pathway to induce non-specific gene silencing via interactions within the 3' UTR. Seed region filters and seed frequency analysis are incorporated in the SMARTselection design strategy to reduce microrna-like off-target activity. Conserved microrna seed regions are filtered out of designs Frequency analysis of seed complements (how often this occurs in the 3' UTR transcriptome) are performed and preference is given to designs with low-frequency seeds 1 Build Filter Identify target gene Score Crop BLAST Pick Synthesize # Off-Target Genes Low Medium High Seed Frequency Class seed region of antisense guide strand (positions 2-7) 5' 3' Antisense strand 3' 5' Sense strand s with lower seed complement frequency induce fewer off-targets 3. Five s with low, medium or high frequency seed regions were transfected into HeLa cells and their associated off-target signatures assessed using global expression profiling (Agilent 22K platform). sequences were constant at positions 1 and 8-19; only the seed regions positions (2-7) were altered. Low frequency seeds: < 35 occurrences in the HeLa transcriptome. Medium frequency: occurrences. High frequency: > 38 occurrences (RefSeq15). Figure adapted from Anderson et al. (28), see published citation 3 for full experimental details. References 1. Reynolds, A. et al. (24) Rational design for RNA interference. Nature Biotechnology 22(3): Birmingham, A. et al. (26) 3 -UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nature Methods 3(3): Anderson, E. et al. (28) Experimental validation of the importance of seed complement frequency to specificity. RNA 14(5): Technologies Technologies 19

12 L-RAT-XX Functional data Studies have shown that off-target effects are primarily driven by antisense strand seed activity. Therefore, modifications that only enhance antisense strand uptake may increase the number of off-targets caused by the antisense strand2. The ON-TARGETplus modification pattern combines sense strand inactivation with a novel seed region modification. This patented, combined approach is the best strategy to prevent off-target effects caused by both the sense and antisense strands. Sense strand is modified to prevent uptake by RISC and favor antisense strand loading Antisense strand is modified to destabilize off-target activity and enhance target specificity LQ-MOUSE-XX LQ-RAT-XX LU-HUMAN-XX LU-MOUSE-XX LU-RAT-XX J-HUMAN-XX J-MOUSE-XX J-RAT-XX modified for sense strand inactivation CDH1 ON-TARGETplus modified Related products GSK3B 5X Buffer See p. 64 RNase-free Water See p. 64 HSPE1 DharmaFECT Transfection Reagents See p. 6 ERBB4 ON-TARGETplus Controls See p. 56 Pre-defined and Custom Libraries See p. 68 PPARA # Off-Target Genes 5 6 Unmodified Unmodified SMARTpool ON-TARGETplus 12 Unmodified ON-TARGETplus #2 #3 #2 #2 #3 #3 #1 #4 #1 #1 #4 #4 #2 #3 #2 #2 #3 #3 #1 #4 #2 #2 #3 #3 #1 #1 #4 #4 #1 #1 #4 #4 #1 5 A KT2 2 nm 1 nm 2 nm ITGB1BP1 5 nm 1 nm 2 nm GSK3A 5 nm 1 nm 2 nm 1 nm 2 nm STK39 Target Gene 5 nm 1 nm 2 nm A KA P11 5 nm 1 nm 2 nm MA PK3 5 nm 1 nm 2 nm TESK1 5 nm 1 nm 2 nm PTPRJ 5 nm 1 nm 2 nm PIK4CA 5 nm 1 nm 25 STK17A The ON-TARGETplus SMARTpool reagents are highly potent at low concentrations. Target mrna knockdown with sigenome and ON-TARGETplus SMARTpool is effective down to concentrations of 2 nm. References 1. Jackson, A.L. et al. (26) Position-specific chemical modification increases specificity of -mediated gene silencing. RNA 12(7): Chen, P.Y. et al. (28) Strand specific 5 O methylation of duplexes controls guide strand selection and targeting specificity. RNA 2: Technologies 4 Individual SMARTpool ON-TARGETplus Individual ON-TARGETplus SMARTpool ON-TARGETplus modifications reduce the overall number of off-targets and pooling reduces them even further. Panels (A) and (B) are representative examples of off-target signatures with and without application of ON-TARGETplus modifications to (A) a single and (B) a SMARTpool reagent. Panel C represents quantification of off-target genes downregulated by two-fold or more. Compilation of off-targets induced by the indicated 1 reagents (4 s per gene or a single SMARTpool reagent) were quantified using microarray analysis (Agilent). Green bars indicate genes with 2-fold or more reduction of expression when treated with the indicated reagent. Each shaded box represents the middle 5% of the data set. Horizontal line in box: Median value of the data set. Vertical bars: minimum and maximum data values. #2 #3 #4 sigenome ON-TARGETplus 75 6 ON-TARGETplus SMARTpool 1 5 nm Relative target expression (%) The ON-TARGETplus modification pattern dramatically reduces off-targets. Off-target effects induced by s targeting the indicated genes were quantified using genomewide microarray analysis. For each target, three different pools were used: unmodified (lightest blue), sense strand-inactivated (light blue) and ON-TARGETplus modified (dark blue). Data shown represents the number of genes down-regulated by two-fold or more. HEK293 cells were transfected with 1 nm pool using.2 μl of DharmaFECT 1 and incubated for 24 hours. Unmodified 8 2 ARPC1B mrna Levels gene % gene expression, % geneexpression, expression, % (%) gene expression, % gene expression, gene expression, %% LQ-HUMAN-XX * These are agnostic product identifiers. Actual catalog numbers are gene and species-specific (e.g., L-1257-) ** Minimum purchase of four individuals required for 2 nmol quantity 2 # Off-Target Genes L-MOUSE-XX Individual ; 2**, 5, 1, 2, 5 nmol 1 Blocks sense-strand entry into RISC and improves antisense-strand specificity to its target with a unique seed-region modification1 Delivers optimal potency while eliminating off-targets that could cause false positives Pooling ON-TARGETplus-modified s results in the most specific gene targeting reagent L-HUMAN-XX Set of 4 Upgrade; 2, 5, 1, 2 nmol C 12 ON-TARGETplus Set of 4 ; 2, 5, 1, 2 nmol B A ON-TARGETplus incorporates design strategies for functionality and specificity, and is the only available with a patented dual-strand modification pattern to reduce off targets caused by both strands of the. Catalog No.* SMARTpool reagent; 5, 1, 2, 5 nmol 1 5 nm Technologies I.2 SMARTpool, Set of 4 and individual s Libraries: pre-defined and Cherry-pick Controls: non-targeting and positive 4 I.2 Technologies ON-TARGETplus SMARTpool SMARTpool SMARTpool SMARTpool SMARTpool SMARTpool SMARTpool SMARTpool SMARTpool Control False phenotypes are alleviated by ON-TARGETplus SMARTpool reagents while target gene knockdown is maintained. The effect of silencing ARPC1B on cell migration was studied in a breast cancer cell line. A monolayer of cells was uniformly scraped and the rate of cell migration to close the scrape (wound healing) was evaluated. Both unmodified and ON-TARGETplus reagents induced potent target knockdown. Inconsistent phenotypes due to off-target effects (red outline), were observed for cells transfected with unmodified individual s. The unmodified SMARTpool improved the false phenotype considerably, while the ON-TARGETplus SMARTpool significantly reduced off-target effects to produce a consistent phenotype. Unmodified In collaboration with Kaylene Simpson, Laura Selfors and Joan Brugge, Harvard Medical School. ON-TARGETplus Technologies 21

13 Technologies I.2 Accell SMARTpool, Set of 4 and individual s Libraries: pre-defined and Cherry-pick Controls: non-targeting, positive and transfection Accell provides delivery into difficult-to-transfect cell types for unprecedented experimental flexibility and discovery. 4 1 Easy to use for rapid, high confidence results Accell reagents are specially modified for uptake without a separate delivery reagent - no complexing wait time, no wash steps. Simply combine Accell with optimized Accell Delivery Media and add to cells. Accell (stock solution) A Accell Delivery Media I.2 Technologies Accell Pre-designed SMARTpool reagent; 5, 1, 2, 5 nmol Set of 4 ; 2, 5, 1, 2 nmol Set of 4 Upgrade; 2, 5, 1, 2 nmol Catalog No.* E-HUMAN-XX E-MOUSE-XX E-RAT-XX EQ-HUMAN-XX EQ-MOUSE-XX EQ-RAT-XX EU-HUMAN-XX EU-MOUSE-XX EU-RAT-XX Deliver without the need for transfection reagents, instruments or viral vectors Patent-pending novel modifications facilitate uptake, stability, specificity and knockdown efficiency Proven performance in neuronal, immunological, primary and other difficult-to-transfect cell types Appropriate for use in vivo, due to stabilizing modifications Extended-duration knockdown with optimized continuous application Functional data Accell achieves what no other RNAi product can claim: delivery into difficult-to-transfect cells without additional reagents, virus or electroporation. Researchers around the world are achieving targeted gene silencing in cells that had previously been beyond the reach of conventional RNAi products due to toxicity caused by transfection reagents or undesirable viral responses. The Accell delivery protocol simplifies targeted gene knockdown. (A) Combine 1 µm Accell and Accell Delivery Media or other low- or no-serum media. (B) Add Accell delivery mix directly to cells and incubate for 72 hours. B Accell delivery mix (1 µm ) A B C D E F G H Individual ; 5, 1, 2, 5 nmol A-HUMAN-XX A-MOUSE-XX A-RAT-XX * These are agnostic product identifiers. Actual catalog numbers are gene and species-specific (e.g., E-1257-) White matter Granular layer Purkinje layer Molecular layer Molecular layer Purkinje layer Peer-reviewed publications demonstrate effective silencing in numerous cell types ARPE-19 (human retinal epithelial cells) BxPC3 (pancreatic tumor cell lines) β-islet cells Bone marrow cells C1 tumor derived cells Bronchial smooth muscle cells (BSMC) Caco-2 (colon colorectal adenocarcinoma) Cardiomyocytes Related products 5X Buffer See p. 64 Accell Delivery Media See p. 64 RNase-free Water See p. 64 Accell Controls See p. 56 Pre-defined and Custom Libraries See p. 68 A Granular layer Accell demonstrates effective uptake and silencing in organotypic brain slices. Accell shows increased uptake with extended incubation. 25 μm cerebellar sections were prepared, cultured and incubated for 3 hours (A) and 72 hours (B) with Accell Red Non-targeting control (NTC) before inspection by microscopy. B CD4+ primary human T cells CD14+ primary monocytes GH3 (rat somatolactotrophs pituitary cell line) H9 stem cell lines HCT-116 (colorectal carcinoma) HUVEC JJN3 (plasma cell leukemia) MEC1 (human chronic B cell leukemia) MN-1 MS1 (mouse pancreatic islet endothelia cells) NOD CD4+CD25 splenic cells Cerebellar granule neurons (CGN) Cortical neurons Endometrial cells Endothelial cells Extravillous trophoblasts (EVT) Hepatocytes Immortalized B cells Keratinocytes Lymphocytes Macrophages Mantle cell lymphoma cells (MCL) Over 7 peer-reviewed publications cite the success of Accell on gelifesciences.com/ accell-citations NOXA Monocytes OVCA 42 (ovarian carcinoma) Mouse embryonic fibroblasts (MEF Media only 96 hours NTC 48 hours NTC 72 hours NTC 96 hours Accell PPIB 48 hours Accell PPIB 72 hours Accell PPIB 96 hours PGA-1 (lymphocyctic leukemia B cell line) RAW264.7 macrophages SHSY5Y (neuroblastoma) Natural killer (NK) cell line Neurons (primary rat) Oligodendrocyte precursors Accell has special experimental considerations. Learn more on page 23 anti- Cyclophilin B (PPIB) anti-actin anti-gapdh NTC 72 hours NTC 96 hours Accell GAPDH 48 hours Accell GAPDH 72 hours Accell GAPDH 92 hours Accell effectively silences target genes in cultured P8 mouse brain slices. Cultured brain slices were assessed following incubation with Accell targeting Cyclophilin B (PPIB) and GAPDH or Non-targeting control (NTC) at three time points (48, 72, 96 hours) and the level of remaining protein was evaluated using western blot analysis (Actin as loading control). SNB19 glioma cells T47D (ductal breast epithelial tumor cell line) T98 glioma cells THP-1 monocytes U266 (peripheral blood B lymphocyte myeloma) U937 (leukemic monocyte lymphoma) Pancreatic tumor cell lines Peripheral blood mononuclear cells (PBMC) Vascular smooth muscle cells (VSMC) Experimental considerations: Accell requires a higher concentration than conventional ; be sure to order enough for your experiments ( see p. 25 for more details). Accell is recommended for use with Accell Delivery Media (Cat #B-5). This is a serum-free media specially formulated and optimized for use with Accell. It has undergone thorough testing and successful validation in over 4 cell lines. Other serum free/low serum media have also been used successfully with Accell. See protocol for details. Delivery is inhibited by the presence of BSA in serum. Serum-free media formulations or < 3% serum in standard media is recommended. Full-serum media can be added back as early as 48 hours after transfection, but optimal mrna silencing is typically achieved by 72 hours. Technologies Technologies 22 23

14 Technologies I.2 sigenome SMARTpool, Set of 4 and individual s Libraries: pre-defined and Cherry-pick Controls: non-targeting and positive Catalog No.* sigenome Pre-designed SMARTpool reagent; 5, 1, 2, 5 nmol Set of 4 ; 2, 5, 1, 2 nmol Set of 4 Upgrade; 2, 5, 1, 2 nmol Individual ; 2**, 5, 1, 2, 5 nmol Related products M-HUMAN-XX M-MOUSE-XX M-RAT-XX MQ-HUMAN-XX MQ-MOUSE-XX MQ-RAT-XX MU-HUMAN-XX MU-MOUSE-XX MU-RAT-XX D-HUMAN-XX D-MOUSE-XX D-RAT-XX * These are agnostic product identifiers. Actual catalog numbers are gene and species-specific (e.g., M-1257-) ** Minimum purchase of four individuals required for 2 nmol quantity 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 sigenome Controls See p. 56 Pre-defined and Custom Libraries See p. 68 sigenome s are highly potent and reliable gene-silencing reagents. Since its launch in 24, sigenome has provided the longest standing guarantee on the market. sigenome designs incorporate rational strand bias with application of ON-TARGET modifications when required for optimal functionality Trusted in more whole genome screens than any other Functional data In addition to identifying good designs, the creation of sigenome s also involves: Thermodynamic analysis to determine optimal antisense strand-loading characteristics for effective silencing BLAST analysis of both strands to alleviate cross-targeting To retain more high-scoring designs, the above analysis may trigger the use of a sense strandblocking modification (ON-TARGET) to ensure only the appropriate strand enters RISC for effective target knockdown. Target mrna Levels (%) MAP2K1 sigenome SMARTpool reagents are potent at 5 nm working concentration. A comparison of target gene silencing at 5 and 1 nm concentration illustrates the high potency of sigenome SMARTpool reagents. 5 or 1 nm sigenome SMARTpool reagents targeting the indicated genes were transfected into HeLa cells (1 cells/well) using.1 µl/well DharmaFECT 1 Transfection Reagent. The negative control (NTC pool) was sigenome Non-targeting Pool #2. 1 nm concentration 5 nm concentration VIM SPTN2 RB1 PPPIRII AURKB ARPC3 LATS1 CDC42 ARPC1B MBIP ACTB ALDOA CAMK2G DNMT1 Target Gene EPHA7 PPIB PRKCA FLNA 4 1 LMNA NFIA MAPK6 GAPDH DBI NEK7 CAMKK2 NTCpool Limit freeze-thawing of s to 5 events ordering guide Use our online calculator to determine how much and transfection reagent you need for your experiment. View the Calculate Quantities tab at gelifesciences.com/dharmacon/sirna-calculators/ Both ON-TARGETplus and sigenome reagents are routinely used at 5-2 nm concentrations. The calculations are based on 25 nm for estimation purposes only. Due to the unique nature of Accell delivery, it requires a higher concentration than conventional s. The table reflects the recommended 1 μm working concentration. ON-TARGETplus and sigenome Approximate number of reactions (wells) at 25 nm concentration nmol 96-well plate 24-well plate 12-well plate , , 8 4 Accell Approximate number of reactions (wells) at 1 μm concentration nmol 96-well plate 24-well plate 12-well plate Estimated numbers are provided for your convenience in ordering and do not account for pipetting error. I.2 Technologies Target mrna Levels (%) HeLa MCF1a Target Gene >9% knockdown 8-89% knockdown 75-79% knockdown 7-74% knockdown <7% knockdown sigenome SMARTpool reagents provide effective, high confidence gene silencing in screens. HeLa and MCF 1A cells were transfected with 5 nm each across 44 Kinase genes using DharmaFECT 1 Transfection Reagent. Target microrna levels were determined by branched DNA assay (Panomics Quantigene Reagent System). The pie chart represents the summary of silencing achieved for the entire data set (88 data points). The bar graph is a representative sample of genes assayed. knockdown guarantee sigenome and ON-TARGETplus reagents (SMARTpool and at least three of four individual s) are guaranteed to silence target gene expression by at least 75% at the mrna level when used under optimal delivery conditions (confirmed using validated positive and negative control s). Silencing should be monitored at the mrna level hours after transfection using up to 1 nm of. 24 Technologies Technologies 25

15 Technologies I.2 Lincode for lncrna SMARTpool, Set of 4 and Individual s Libraries: pre-defined and Cherry-pick Controls: Non-targeting and positive Catalog No.* Lincode pre-designed SMARTpool R-HUMAN-XX reagent; 5, 1, 2, 5 nmol R-MOUSE-XX Set of 4 Upgrade; RU-HUMAN-XX 2, 5, 1, 2 nmol RU-MOUSE-XX Individual ; N-HUMAN-XX 2 **, 5, 1, 2, 5 nmol N-MOUSE-XX * These are agnostic product identifiers. Actual catalog numbers are gene and species-specific (e.g., R ) ** Minimum purchase of four individuals required for 2 nmol quantity Related products Lincode s have been created to support the growing interest in analysis of long noncoding RNAs (lncrna). As with sigenome and ON-TARGETplus s, which target protein-coding genes, Lincode s are designed with the SMARTselection algorithm to ensure high-efficiency silencing. Lincode s also carry the proprietary ON-TARGETplus dual-strand chemical modifications to ensure optimal strand loading and disrupt microrna-like seed activity for the reduction of off targets. Pre-designed against characterized lncrnas (NR_ and XR_ transcripts) in Human RefSeq database Lincode s carry proprietary dual-strand chemical modifications to ensure optimal strand loading for the reduction of off-targets SMARTpool reagent technology targets multiple regions of the lncrna to improve the likelihood of RNAi-mediated degradation Functional data The Lincode product line targets human noncoding RNA genes in the RefSeq database that meet the following criteria: 2 nt in length Gene record contains at least one RefSeq Accession prefix of NR_ or XR_ RNA type "non-coding RNA" or "miscellaneous RNA" to exclude known classes like trna, rrna, etc. microrna, another type of non-coding RNA, is modulated by reagents available in the miridian product line) Cherry-pick TM RNAi Libraries put the control in your hands! Enjoy fast and easy online ordering - no quote required! Build your own custom RNAi library with our online Cherry-pick RNAi Library Plater. It offers the flexibility to configure a library of and microrna reagents for your unique individual experimental needs Select ALL of the genes of interest Decide which RNAi products and controls best meet your needs Combine multiple product lines, formats, and species Choose the quantity required for your experimental goals When Dharmacon pre-defined RNAi Libraries don't match your experimental requirements, you can make your own custom library with ease, speed and economy. Just like our pre-defined libraries, Cherry-pick Libraries are made up of our premier and microrna product lines for human and mouse. Here's how: I.2 Technologies 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 Lincode Controls See p. 57 Pre-defined and Cherry-pick Libraries See p. 68 Percent Expression A 18% 16% 14% 12% 1% 8% 6% 4% 2% 25 nm Viability 14% 12% 1% 8% 6% 4% 2% Viability 1. Load gene identifiers for your desired and microrna products into our online Cherry-Pick Library Plater. Go to cherry-pick-libraries NCBI Gene ID or Gene Symbol mirbase mature name or accession Dharmacon product catalog numbers 2. Review the results for accuracy and product availability B Percent Expression % SMARTpool CDKN2B-AS1 25 nm Viability 14% 12% 1% 8% 6% 4% 2% % SMARTpool Untreated % 14% 12% 1% 8% 6% 4% 2% % Viability Detection of effective lncrna knockdown following application of Lincode reagents. (A) Knockdown of CDKN2B- AS1 in HeLa cells. (B) Knockdown of BDNF-AS1 in hndf cells. All s were used at 25 nm. lncrna transcripts were detected with Solaris qpcr lncrna Expression Assays and normalized to Non-targeting Control. Viability was assessed by resazurin assay. 1 Load Identifiers 2 Review Results 3 Select Products 3. Select the product lines and product formats to include on the plates 4. Configure the library plates for quantity, controls, replicates and layout Then simply add to your cart! 4 Configure Plates BDNF-AS1 Untreated 26 Technologies Technologies 27

16 shrna Technologies I.3 I.3 shrna Technologies Which shrna reagent is best for you? shrna Technologies The industry s broadest, most technologically advanced portfolio of shrna reagents for transient, long-term, inducible and in vivo RNA interference. SMARTchoice shrna GIPZ TRIPZ TRC Choice of 7 constitutive and 4 inducible promoters Human CMV Pol II TRE min-cmv U6 Pol III Lentiviral Lentiviral Lentiviral Lentiviral GFP RFP Species Promoter Vector backbone Fluorescent reporter gene GFP RFP Guaranteed silencing* Achieve specific knockdown with minimal toxicity Perform transient or long-term gene silencing, even in difficult-totransfect cell types In vivo RNAi Create stable cell lines Recommended for primary and non-dividing cells Inducible expression pages pages 3-31 Whole genome library availability Formats Learn more on page High-titer purified lentiviral particles for efficient transduction Viral particles are shipped on dry ice Study essential gene function with inducible shrna Follow the icons to your product solution page 39 page 34 and 36 Table of Contents shrna tools selection guide Less toxicity, more specificity with microrna-adapted shrna... 3 SMARTchoice Lentiviral shrna SMARTchoice Inducible Lentiviral shrna GIPZ Lentiviral shrna High-titer viral particles Transduction-ready concentrated lentiviral particles supplied in tubes. Bacterial glycerol stock Bacterial culture transformed with a plasmid vector, grown in the presence of glycerol for freezer storage and supplied in single tubes. Available as individual clones, target gene sets (at least three constructs per target gene) and pre-defined or custom libraries. Arrayed library format Genome-scale, pre-defined or custom libraries of bacterial glycerol stocks arrayed in 96-well microtiter plates. Gene families and pathways Pre-defined libraries arranged by gene family and/or function and arrayed in 96-well microtiter plates. s hrna Starter Kit All reagents and protocols necessary for starting a gene silencing experiment are provided in a single, convenient package ( see p. 36 for more details). GFP RFP Fluorescent markers Visually track expressed shrna with TurboGFPTM (green fluorescent protein) or TurboRFPTM (red fluorescent protein). Species Human TRIPZ Inducible Lentiviral shrna Mouse Rat TRC Lentiviral shrna GIPZ and TRIPZ shrna Starter Kits High-titer lentiviral particle production shrna Technologies 28 * For GIPZ and TRIPZ shrna, at least one out of three shrna constructs is guaranteed to reduce target mrna levels by 7% or more when used in combination with the non-targeting control and the GAPDH positive control. shrna Technologies 29

17 shrna Technologies I.3 Less toxicity, more specificity with microrna-adapted shrna Harnessing the endogenous microrna pathway I.3 shrna Technologies microrna-adapted shrnas offer a clear advantage over simple hairpin shrnas. All shrna designs embed the gene-specific silencing sequence in a primary microrna transcript, thus creating a RNAi trigger that silences genes with increased specificity. microrna-adapted shrna Nucleus (1) (2) Extra-chromosomal expression from plasmid, or genomically integrated expression from lentivirally delivered sequence microrna-based shrnas exhibit lower cellular toxicity and off-target effects Microprocessor (Drosha/DGCR8) Simple hairpin shrna Exportin-5 Simple stem-loop designed hairpins driven by strong promoters may cause cellular toxicity. Reducing the multiplicity of infection (MOI) can be insufficient to decrease the toxic effects potentially caused by the accumulation of transcripts. Published findings show a significant reduction in neurotoxicity could be attributed to moving the silencing sequence from a simple hairpin to a microrna scaffold. 1 Cytoplasm Mature microrna duplex Dicer complex Simple stem-loop hairpins may disrupt normal microrna processing and function, causing cell death and off-target effects. Published findings suggest that the U6 promoter may interact with certain cancer-causing genes to accelerate tumorigenesis and cause toxicity. 2 Mature microrna RISC Off-target silencing resulting in measurable phenotypic effects were observed in vitro following transduction of simple stem-loop hairpin shrna for several genes tested. In addition, persistent disruption of microrna biogenesis was observed for six critical cellular micrornas, causing 6-8% suppression of native micrornas and up to threefold up-regulation of putative target genes. 3 Catalytic mrna cleavage and gene silencing References 1. McBride, J.L et al. (28) Artificial micrornas mitigate shrna mediated toxicity in the brain: Implications for the therapeutic development of RNAi. PNAS 15(15): Beer, S. et al. (21) Low-level shrna cytotoxicity can contribute to MYC induced hepatocellular carcinoma in adult mice. Molecular Therapy 18(1): Pan, Q. et al. (211) Disturbance of the microrna pathway by commonly used lentiviral shrna libraries limits the application for screening host factors involved in hepatitis C virus infection. FEBS Letters 585: Borrowing the endogenous microrna pathway to induce RNAi. shrna approaches include the introduction of genetically engineered viral vectors or plasmid-based vectors expressing silencing sequences embedded in an endogenous microrna scaffold (1) or simple stem-loop shrna (2). Expressed sequences (1 and 2, shown in blue) enter the endogenous pathway at an early stage and are efficiently processed into potent silencing molecules using the endogenous microrna mechanism ( see p. 9). All of these approaches lead to target mrna cleavage (shown in purple) and gene silencing. 3 shrna Technologies shrna Technologies 31

18 Ψ Ψ 5ʹ LTR 5' LTR.6 SV4 ori IRES PuroR Ψ.2 ptripz AmpR MOI 2 puc ori MOI 5 hcmv GAPDH tgfp IRES PuroR MOI 2 R WPRE shrna Ψ RRE R SV4 ori MOI 1 SV4 ori AmpR shrna puc ori RRE hpgk shrna PuroR Ψ Ψ PuroR RSV/5ʹ LTR RSV/5' LTR AmpR plko.1 Researcher Experiment Dharmacon Process puc ori AmpR hcmv hcmv RRE IRES microrna PuroR WPRE RRE.5.25 TU ( 15) = hcmv hcmv hcmv mcmv mcmv D CAG mef1α CAG PGK. PGK. UBC UBC UBC HEK293T Jurkat Make informed decisions in the design of gene silencing experiments using the SMARTchoice shrna Promoter Selection Plate. Human A549, HEK293T and Jurkat cells were transduced with lentiviral particles arrayed in the SMARTchoice shrna Promoter Selection Plate. TurboGFP expression was assessed by fluorescent microscopy 72 hours post-transduction. Images clearly demonstrate that the most functional promoter in the A549 cells is mcmv, whereas the hcmv promoter is most active in HEK293T, and the mef1α promoter is most active in Jurkat cells. Serial Dilutions.5.25 tgfpnuc -2a- Blast R ORF RRE WPRE H ploc Promoter A Empty B hcmv: human cytomegalovirus C mcmv: mouse cytomegalovirus D hef1α: human elongation factor 1 alpha E mef1α: mouse elongation factor 1 alpha F CAG: chicken β actin hybrid promoter G PGK: mouse phosphoglycerate kinase H UBC: human ubiquitin C 4. shrna vector construction SMARTchoice promoter and fluorescent reporter of choice tgfpnuc -2a- BlastR WPRE ploc SV4 ori puc ori IRES Multi Tag Cloning Site AmpR hef1α PGK. TU ( 15) = G R puc ori SV4 ori hcmv mcmv hef1α mef1α CAG PGK TU ( 1 ) = 5 SMARTchoice shrna Empty hcmv UBC 5' LTR Ψ 5. Lentiviral particle production High-titer particles Transduction-ready tgfp RRE or trfp IRES PuroR WPRE SMARTvector universal scaffold 3' SIN LTR 5ʹ LTR Ψ RRE mcmv hef1α mef1α CAG hcmv mcmv hef1α mef1α CAG PGK UBC tgfp or trfp 2. IRES PuroR Select optimal promoter Based on fluorescence intensity WPRE 3ʹ SIN LTR SMARTvector2. universal scaffold PGK. UBC SMARTchoice shrna 6. Perform gene silencing experiments Utilizing matched controls 1.4 Relative Expression (Normalized to NTC) mef1α CAG F PGK UBC SMARTchoice promoters mef1α hef1α Promoter hef1α CAG Multi Tag Cloning Site AmpR A549 The SMARTchoice shrna Promoter Selection Plate enables straightforward qualitative assessment of promoters that actively drive expression. After transduction of cells, wells can be quickly and visually evaluated for TurboGFP intensity using fluorescence microscopy, high-content imaging or detection using a microplate reader. hcmvhef1α ORF mef1α IRES E Empty mcmv Promoter Promoter 1. WPRE 3ʹ SIN LTR SMARTchoice shrna Controls See p C 3' SIN LTR Related products 4. Empty B mcmv SMARTchoice promoters TU ( 15) = 8. microrna 2. Transduce cells of interest Amp puc ori SV4 ori Cells specific to your application hcmv Promoter.25 PuroR psmart 2. Ψ.5 A hcmv.5.25 SV4 ori 5ʹ LTR 1. Promoter puc ori Empty 5ʹ LTR Ψ 5' LTR 2. AmpR Ψ 4. Empty Serial Dilutions psmart 2. TU ( 15) = ' LTR TU ( 15) = 8. Serial Dilutions IRES 3ʹ SIN LTR Serial Dilutions Serial Dilutions tgfp Ψ tgfp 3' SIN LTR 1. SMARTchoice Promoter Selection Plate Arrayed viral particles Normalized titers 3ʹ SIN LTR 3' SIN LTR plko.1 Functional data ** Estimated manufacturing time is four to six weeks. SV4 ori U6 hpgk U6 RRE 5ʹ LTR 5' LTR puc ori 3ʹ SIN LTR psmart 2. AmpR PuroR R WPRE Ψ rtta3 shrna 5ʹ LTR.4. trfp IRES R UBC TRE RRE 5' LTR Relative Expression (Normalized to NTC) puc ori.8 puc ori * These are agnostic product identifiers. Actual catalog numbers are gene and species-specific (e.g., SH ) shrna Technologies 1. tgfp shrna 3' SIN LTR Set of 3 Lentiviral SKXX-HUMAN-XX Particles; SKXX-MOUSE-XX 1 or 2 µl SKXX-RAT-XX 1x18 TU/mL pgipz WPRE Relative gene silencing of NIH/3T3 cells transduced with shrna expressed from pgipz mcmv and hcmv promoters Amp puc ori SV4 oriidentifying the optimal promoter for your cells of interest using the SMARTchoice UBC TRE Promoter Selection Plate allows for RRE trfp rtta3 IRES Puro WPRE increased shrna functionality and shrna performance - resulting in successful ptripz knockdown of your target gene in cells Amp puc ori SV4 oriof interest, including rodent, primary, MOI 1 MOI 5 embryonic stem and neuronal cells. hcmv Selection of the mcmv promoter using the RHOA-1 RRE cppt/cts tgfp IRES Puro shrna WPRE SMARTchoice Promoter Selection Plate psmart 2. in mouse NIH/3T3 cells results in greater knockdown of targeted gene expression. RRE WPRE shrna AmpR SMARTchoice shrna Promoter Selection Plate allows straightforward identification of optimal promoters in cells of interest Custom SMARTchoice vectors based on advanced microrna-adapted shrna design for specific silencing and minimal off-target effects Flexibility to order gene-specific shrna vectors with choice of seven different promoters and two fluorescent reporters to maximize success SHXX-HUMAN-XX Individual Lentiviral Particles; SHXX-MOUSE-XX 1 or 2 µl SHXX-RAT-XX SMARTchoice Lentiviral shrna PuroR Ψ SP-1-1 IRES 3ʹ SIN LTR Catalog No.* tgfp 3' SIN LTR To fully harness the utility of lentiviral vector approaches in shrna-mediated gene silencing, careful attention must be paid to the choice of promoter controlling its expression. Inefficient promoter activity due to varying cellular and biological contexts results in sub-optimal knockdown and can be misinterpreted as poor shrna functionality. The SMARTchoice shrna platform permits the researcher to first observe activity of multiple promoters in the target cells of interest and then order SMARTvector gene-specific lentiviral shrna and controls with optimal promoter choices, saving time and money by making informed decisions. hcmv hcmv mcmvrre hcmv 1.4 SMARTchoice shrna Promoter Selection Plate Custom manufactured high-titer lentiviral particles** SMARTchoice Promoter Selection Plate RFP 3ʹ SIN LTR GFP 3' SIN LTR shrna Technologies I.3 SMARTchoiceTM Lentiviral shrna From PowerPoint (R&D) I.3 shrna Technologies Revised versions MOI 2 MOI 1 MOI 5 GAPDH SMARTchoice experimental workflow. Identify the ideal promoter for your cells of interest with the transduction-ready SMARTchoice shrna Promoter Selection Plate. Next, place an online order for SMARTchoice Lentiviral shrna particles with your choice of promoter and reporter and save the extensive time, labor and money required for production of high-titer lentiviral particles in your lab. Benefit from our internal quality-controlled process of cloning and packaging your customized vector into lentiviral particles using packaging technology for enhanced biosafety. shrna Technologies 33

19 BRCA1-1 BRCA1-2 BRCA1-3 BUB1-1 BUB1-2 BUB1-3 MAPK1-1 MAPK1-2 MAPK1-3 MAPKAPK2-1 MAPKAPK2-2 MAPKAPK2-3 PTEN-1 PTEN-2 PTEN-3 TP53-1 TP53-2 TP53-3 shrna Technologies I.3 34 SMARTchoice TM Inducible Lentiviral shrna Individual shrnas and sets of three Controls: non-targeting and positive (PPIB and GAPDH) Use the SMARTchoice Inducible Nontargeting 4-pack to determine the optimal promoter for your experiment Related products SMARTchoice Inducible shrna Controls See p. 57 shrna Technologies Catalog No.* SMARTchoice Inducible Lentiviral shrna Particles Individual VSH* (Human) SMARTchoice Inducible Lentiviral VSM* (Mouse) shrna; 1 or 2μL, 1 7 TU/mL VSR* (Rat) Set of 3 VSH* (Human) SMARTchoice Inducible Lentiviral VSM* (Mouse) shrna; 1 or 2μL, 1 7 TU/mL VSR* (Rat) *These are generic product identifiers. Actual catalog numbers will be gene, species, reporter, and promoter-specific. (e.g., BSH6461-EG434). Estimated manufacturing time is four to six weeks. Combining all of the advantages of the SMARTvector shrna design innovations with the recently developed Tet-On 3G tetracycline-inducible expression system and the flexibility of SMARTchoice promoter and reporter options, this novel single-vector regulatable RNAi system permits the rapid development of stable cells with tightly controlled shrna expression. Advanced universal scaffold and proprietary shrna design algorithm for potent silencing at singlecopy integration and minimal off-target effects Tight control of shrna and reporter gene expression utilizing the latest generation Tet-inducible expression technology, the Tet-On 3G Inducible System Unsurpassed flexibility with your choice of two reporters and four promoters in an inducible system Functional data P TRE3G SMARTchoice Inducible shrna mcmv PGK mef1α hef1α 5' LTR Ψ RRE tgfp Puro R 2a Tet-On 3G or trfp SMARTvector universal scaffold Elements of the SMARTchoice Inducible shrna lentiviral backbone. The SMARTchoice inducible vector customization options include four constitutive promoter and two reporter options. While the inducible control of the SMARTvector shrna and the reporter gene are conferred by the Tet 3G promoter (PTRE3G), the expression of the Tet-On 3G transactivator protein is under the control of a constitutive RNA pol II promoter. The SMARTchoice Inducible shrna constitutive RNA pol II promoter options include: mcmv (mouse cytomegalovirus intermediate early promoter); PGK (mouse phosphoglycerate kinase promoter); hef1α (human elongation factor 1 alpha promoter); and mef1α (mouse elongation factor 1 alpha promoter). Fluorescent reporter options are TurboGFP and TurboRFP. A Cell Proliferation B U2OS cells, SMARTchoice shrna with mcmv C 1 (U2OS, mcmv promoter) 9 8 No Dox 5 ng/ml Dox 5 ng/ml Dox NTC UBB Relative Cell Number Days in Culture NTC No Dox NTC 5 ng/ml Dox NTC 5 ng/ml Dox UBB No Dox UBB 5 ng/ml Dox UBB 5 ng/ml Dox SMARTchoice Inducible shrnas enable tightly controlled knockdown of essential genes in a time- and doxycycline dose-dependent manner. (A) U2OS cells were transduced at MOI =.1 with SMARTchoice Inducible mcmv vectors carrying either a non-targeting control shrna (NTC) or an shrna directed against the ubiquitin B (UBB) gene. Cells were selected for 3 days with 1.5 µg/ml puromycin. After selection, cells were seeded at 2 cells per/well in 96-well plates. 24 hours later (Day 1), shrna expression was induced with the indicated dose of doxycycline, and cell number was then measured each day with the Cell Titer-Glo assay (Promega). Each data point represents the mean and standard deviation of six independent wells. (B) On Day 6, following 5 days of exposure to doxycycline, the cells were stained with Hoescht and cell nuclei (blue) and TurboGFP (green) were imaged on the Thermo Scientific ArrayScan VTI HCS Reader. (C) Cell density, defined as number of nuclei per field was computed for 18 fields per condition. SMARTchoice promoters Cells per Field WPRE 3' SIN LTR GFP Terminal Cell Density (U2OS, mcmv promoter) NTC UBB RFP GIPZ Lentiviral shrna Perform specific and efficient, transient or long-term RNAi with GIPZ Lentiviral shrna. Human and mouse genes are each targeted with multiple shrna constructs that have been cloned into the pgipz lentiviral vector. Efficient and guaranteed* knockdown TurboGFP tracks shrna expression Lentiviral delivery extends RNAi to primary and non-dividing cells Bacterial glycerol stocks or purified, high-titer lentiviral particle format** Functional data Remaining mrna Expression (%) % 39 % Ψ 5' LTR RRE 26 % 26 % hcmv tgfp Amp R 9 % 11 % 15 % IRES 32 % puc ori Puro R pgipz 4 % 23 % shrna SV4 ori Vector Element Utility hcmv Human cytomegalovirus promoter drives strong transgene expression tgfp TurboGFP reporter for visual tracking of transduction and expression Puro R Puromycin resistance permits antibiotic-selective pressure and propagation of stable integrants IRES Internal ribosomal entry site allows expression of TurboGFP and puromycin resistance genes in a single transcript shrna microrna-adapted shrna (based on mir-3) for gene knockdown 5' LTR 5' long terminal repeat 3' SIN LTR 3' self-inactivating long terminal repeat for increased lentivirus safety Ψ Psi packaging sequence allows viral genome packaging using lentiviral packaging systems RRE Rev response element enhances titer by increasing packaging efficiency of full-length viral genomes WPRE Woodchuck hepatitis posttranscriptional regulatory element enhances transgene expression in the target cells 3 % 117 % WPRE 7 % 8 % 3' SIN LTR Effective knockdown using GIPZ lentiviral shrna. OVCAR-8 cells were transduced with GIPZ lentiviral shrna constructs at MOI =.4-2 in 2-4 biological replicates. Cells were puromycin-selected (3 μg/ml) starting 48 hours posttransduction. RNA was isolated 84 hours post-transduction. qpcr was performed in triplicate via TaqMan Gene Expression Assays using 18S rrna as an internal reference. On average two out of three shrna produced greater than 7% knockdown compared to the GIPZ Non-targeting Control shrna. 12 % 7 % 18 % 25 % Related products GFP Individual shrna clones and target gene sets Individual lentiviral particles and sets of three shrna Starter Kit Libraries: pre-defined and custom Controls: empty vector, non-targeting and positive * At least one out of three constructs is guaranteed to reduce target mrna levels by 7% or more when used in combination with the non-targeting control and the GAPDH positive control. ** Estimated manufacturing time is four to six weeks. TurboFect Transfection Reagent See p. 62 GIPZ and TRIPZ shrna Starter Kits See p. 38 Trans-Lentiviral shrna Packaging Kit See p. 63 GIPZ shrna Controls See p. 57 Pre-defined and Custom shrna Libraries See p. 68 Avoid freezethaw cycles of bacterial glycerol stocks and high-titer viral particles shrna Technologies I.3 shrna Technologies 35

20 shrna Technologies I.3 TRIPZ Inducible Lentiviral shrna Individual clone and target gene set shrna Starter Kit Libraries: pre-defined and custom Controls: empty vector, non-targeting and positive *At least one out of three constructs is guaranteed to reduce target mrna levels by 7% or more when used in combination with the non-targeting control and the GAPDH positive control. The ptripz vector has been engineered to be Tet-On. The tetracycline response element (TRE) fused to the CMV minimal promoter exhibits reduced basal expression and tighter binding to the transactivator. The reverse tetracycline transactivator 3 (rtta3) binds to and activates expression from TRE promoters in the presence of doxycycline. Study the function of essential genes, validate cellular phenotypes and generate regulatable knockdown cell lines with TRIPZ Inducible Lentiviral shrna. Regulatable and reversible gene silencing Guaranteed* knockdown Tet-On or Tet-Off configurations TurboRFP tracks induced shrna expression Functional data 5' LTR Ψ RRE TRE trfp Amp R shrna ptripz puc ori UBC rtta3 IRES SV4 ori Puro R WPRE 3' SIN LTR RFP TRC Lentiviral shrna The RNAi Consortium (TRC) Lentiviral shrna Library was developed by the Broad Institute of MIT and Harvard University. This collection targets both human and mouse genes with broad coverage per transcript. Simple stem-loop shrna design Amenable to in vitro and in vivo applications Lentiviral vector enables transduction of primary and non-dividing cell lines Functional data RSV/5' LTR Ψ RRE puc ori U6 shrna Amp R plko.1 hpgk Puro R 3' SIN LTR Individual shrna clones and target gene sets Libraries: pre-defined and custom Controls: empty vector and positive Related products TurboFect Transfection Reagent See p. 62 Trans-Lentiviral shrna Packaging Kit See p. 63 TRC shrna Controls See p. 57 Pre-defined and Custom shrna Libraries See p. 68 I.3 shrna Technologies 36 Remaining mrna Expression (%) Related products TurboFect Transfection Reagent See p. 62 GIPZ and TRIPZ shrna Starter Kits See p. 38 Trans-Lentiviral shrna Packaging Kit See p. 63 TRIPZ shrna Controls See p. 57 Pre-defined and Custom shrna Libraries See p % NS1 65 % V2THS shrna Technologies 22 % V2THS % V2THS % V2THS % V2THS-1743 V2THS V2THS V2THS TNFRSF12A PRKAR2B DDR1 NS1 PTEN 14 % Vector Element Utility TRE Tetracycline-inducible promoter trfp TurboRFP reporter for visual tracking of transduction and shrna expression shrna microrna-adapted shrna (based on mir-3) for gene knockdown UBC Human ubiquitin C promoter for constitutive expression of rtta3 and puromycin resistance genes rtta3 Reverse tetracycline-transactivator 3 for tetracycline-dependent induction of the TRE promoter Puro R Puromycin resistance permits antibiotic-selective pressure and propagation of stable integrants IRES Internal ribosomal entry site allows expression of rtta3 and puromycin resistance genes in a single transcript 5' LTR 5' long terminal repeat 3' SIN LTR 3' self-inactivating long terminal repeat for increased lentivirus safety Ψ Psi packaging sequence allows viral genome packaging using lentiviral packaging systems RRE Rev response element enhances titer by increasing packaging efficiency of fulllength viral genomes WPRE Woodchuck hepatitis posttranscriptional regulatory element enhances transgene expression in the target cells 35 % 42 % 21 % V2THS % V2THS % 14 % V2THS V2THS Induced knockdown at low MOI. HEK293T cells were transduced with various TRIPZ lentiviral shrna at a MOI of.3, puromycin-selected (5 μg/ml) at 48 hours and induced with doxycycline (1 μg/ml) starting 5 days post-transduction. RNA was isolated 2 weeks post-transduction and knockdown of indicated target genes was compared to the Non-targeting TRIPZ Lentiviral shrna Control (NS1) as determined by TaqMan Gene Expression Assays. Each bar represents an experimental duplicate while qpcr was performed in triplicate using 18S rrna as an internal reference. Vector Element Utility U6 Human U6 (RNA polymerase III) promoter provides high level of expression in the target cells shrna Simple stem-loop shrna for gene knockdown hpgk Human phosphoglycerate kinase promoter drives expression of the puromycin resistance gene Puro R Puromycin resistance permits antibiotic-selective pressure and propagation of stable integrants RSV/5' LTR RSV promoter/5' long terminal repeat promotes strong lentiviral transcription in the packaging cells in the absence of Tat 3' SIN LTR 3' self-inactivating long terminal repeat for increased lentivirus safety Ψ Psi packaging sequence allows viral genome packaging using lentiviral packaging systems RRE Rev response element enhances titer by increasing packaging efficiency of full-length viral genomes Exp 1 Exp 2 Remaining mrna Expression (%) HP1 HP2 HP3 HP4 HP5 shjak2 Gene silencing with TRC lentiviral shrna. Knockdown of six tyrosine kinase genes in A549 lung carcinoma cells with 3 different hairpins performed in duplicate (Exp 1 and 2) and analyzed by qpcr. qpcr reactions were performed with SuperScript II RT Kit and TaqMan Gene Expression Assays. For all six genes, at least one shrna decreased target transcript levels by > 7%. Data adapted from Moffat, et al. (26). 1 HP1 HP2 HP3 HP4 HP5 HP1 HP2 HP3 HP4 HP5 sherbb3 shfgfr1 HP1 HP2 HP3 HP4 HP5 shfyn HP1 HP2 HP3 HP4 HP5 shigf1r References 1. Moffat, J. et al. (26) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high content screen. Cell 124: HP1 HP2 HP3 HP4 HP5 shmst1r shrna Technologies 37

21 shrna Technologies I.3 GIPZ and TRIPZ shrna Starter Kits Catalog No.* GIPZ shrna Lentiviral shrna GIPZ shrna RHS4287 transfection Starter Kit GIPZ shrna transduction Starter Kit RHS586 TRIPZ shrna Lentiviral shrna TRIPZ shrna RHS4741 transfection Starter Kit TRIPZ shrna transduction Starter Kit RHS587 The shrna Starter Kits combine the versatility and specificity of lentiviral shrna, with the simplicity of validated controls and optimized delivery reagents to provide a complete set of reagents for your RNAi experiment with guaranteed silencing. Each kit contains: Multiple human or mouse, GIPZ or TRIPZ shrnas targeting your gene of interest Transfection reagent for optimized shrna delivery (TurboFect for transfection kits or calcium phosphate for transduction kit) Positive and negative shrna controls Trans-Lentiviral Packaging Mix (included in transduction kits only) Guaranteed* silencing High-titer lentiviral particle production Save time and money by transducing your cells of interest with high titer lentiviral particles. Lentiviral particle manufacturing eliminates the substantial investment of time, labor and money required in the design, production and quality control of functional shrna reagents for transduction. All you have to do is order your gene of interest and then add viral particles to cells. SMARTchoice shrna ( p. 35), GIPZ shrna ( p. 42) and Precision LentiORF clones ( p. 87) are available as lentiviral particles. I.3 shrna Technologies * When used with shrna starter kits according to kit instructions. Related products GIPZ Lentiviral shrna See p. 35 TRIPZ Inducible Lentiviral shrna See p. 36 Trans-Lentiviral shrna Packaging Kit See p. 63 5' 3' Bioinformatically identify top target sites to design functional and specific shrnas Clone oligonucleotides into a highly functional scaffold GIPZ and TRIPZ shrna Controls See p. 57 Sequence multiple clones to identify constructs with correct sequence Dharmacon lentiviral shrna design and manufacturing processs shrna knockdown guarantee When you purchase a minimum of three GPIZ or TRIPZ shrnas to the same target, at least one of the shrna constructs will reduce target mrna levels by 7% or more when used with shrna Starter Kits protocols and normalized for delivery efficiency. Transfection conditions should be confirmed using appropriate positive shrna controls provided in the kit, and the percent knockdown should be compared to cells delivered with the non-targeting shrna. Assess gene silencing at mrna and/or protein level Transfect into packaging cells with helper plasmids encoding Gag/Pol and appropriate envelope proteins Isolate viral supernatant and concentrate Transduce into cells of interest All you have to do 38 shrna Technologies shrna Technologies 39

22 microrna Modulation Technologies I.4 Which microrna modulation research tool is best for you? miridian microrna Mimics miridian microrna Hairpin Inhibitors SMARTchoice shmimic Lentiviral micrornas Species Up-regulate or suppress endogenous mature microrna function with rationally designed synthetic and expressed microrna modulation reagents. I.4 microrna Modulation Technologies microrna Modulation Technologies Synthetic modified RNA molecules Promoter Choice of seven promoters Vector Lentiviral Fluorescent reporter gene GFP RFP Chemically modified for improved specificity Modulate microrna levels, even in difficult-to-transfect cell types Achieve specific microrna effects through preferential RISC loading of the correct mature microrna Delivery via transfection reagents Delivery via electroporation Study loss-of-function effects Study gain-of-function effects pages Create stable cell lines page 12 Lentiviral delivery In vivo microrna modulation Whole genome library availability Screen for micrornas that regulate gene expression and affect cellular pathways Specifically express mature micrornas for long-term functional studies page 74 pages FORMATS 1 Learn more on page Follow the icons to your product solution 1 Section I Chapter 4: Table of Contents microrna research tools selection guide... SMARTchoice shmimic Lentiviral micrornas... miridian microrna Mimics... miridian microrna Hairpin Inhibitors Individual microrna: Select individual micrornas in a variety of quantities. High-titer viral particles: Transduction-ready, high-titer lentiviral particle format. Arrayed library format: microrna reagents available in 96-well or 384-well microtiter plates. GFP Fluorescent markers: Visually track expressed microrna with TurboGFP (green fluorescent protein). Species: Human microrna Modulation Technologies 4 Mouse Rat microrna Modulation Technologies 41

23 microrna Modulation Technologies I.4 SMARTchoice shmimic Lentiviral micrornas Individual micrornas as lentiviral particles* Controls: empty vector and negative * Estimated manufacturing time is four to six weeks. Related products shmimic Lentiviral microrna Controls See p. 57 hcmv hef1α mcmv mef1α All images at MOI 3 SMARTchoice shmimic Lentiviral micrornas are ideal for functional analysis of microrna roles through long-term expression of mature microrna. micrornas are endogenous non-coding ~ 22mer RNAs that are highly conserved in mammals and regulate gene expression posttranscriptionally through innate RNA interference mechanisms. As key players in the fine tuning of biological networks, micrornas have significant diagnostic and prognostic potential as biomarkers not only of fundamental cellular physiology but also of disease etiology and progression. To study long-term microrna-induced phenotypes, we have developed a novel system for stable expression of the mature microrna. shmimic lentiviral micrornas are available for each human, mouse and rat microrna described in the mirbase database ( Unique, patent-pending expression scaffold for consistent and correct processing of the mature microrna Universal primary context ensures that each mature microrna is correctly and consistently processed Design modifications promote preferential loading of the mature microrna into RISC and robust function High-titer, concentrated lentiviral particles permits delivery into cells refractory to lipid-based delivery Expression of TurboGFP or TurboRFP (Evrogen, Moscow) allows visualization of transduction efficiency Product Details The SMARTchoice platform includes: The Promoter Selection Plate for straightforward identification of the optimal promoter in the cells of interest Flexibility to order shmimic microrna constructs with a choice of seven different promoters and two fluorescent reporters A HEK293 K562 HUVEC SH-SY5Y B Relative Target mrna Expression Normalized to Matched Negative Control (%) MOI 2 MOI 1 MOI 5 MOI 2 MOI 1 MOI 5 MOI 2 MOI 1 MOI 5 MOI 2 MOI 1 MOI 5 MOI 2 MOI 1 MOI 5 MOI 2 MOI 1 MOI 5 miridian shmimic micrornas induce repression of endogenous human microrna targets in difficult-to-transfect cells. (A) TurboGFP expression in four different cell lines visualized by epifluorescence microscopy 12 hours post-transduction. Puromycin selection was not used in these experiments. (B) HEK293, HUVEC, K562 or SH-SY5Y cells were transduced in biological triplicate with shmimic microrna or negative control viral particles. Cells were harvested for RNA isolation 12 hours post-transduction (no puromycin). Repression of endogenous gene targets was determined using either Thermo Scientific Solaris qpcr Gene Expression Assays and Master Mix (HEK293 and SH-SY5Y data) or Thermo Scientific TM Verso TM 2-Step RT-qPCR SYBR Green Kit (HUVEC and K562 data). Data was normalized to a reference gene (PPIB) and further normalized to matched shmimic negative control. Let-7a HMGA2 mir-429 ZEB1 mir-122 ALDOA Let-7a HMGA2 mir-429 ZEB1 mir-429 ZEB1 HEK293 HUVEC K562 SH-SY5Y I.4 microrna Modulation Technologies TU (x 1 5 ) = Promoter Empty A hcmv B mcmv C hef1α D mef1α E CAG F PGK. G Ubi. H 96-well SMARTchoice Promoter Selection Plate layout. hcmv: human cytomegalovirus; mcmv: mouse cytomegalovirus; hef1α: human elongation factor 1 alpha; mef1α: mouse elongation factor 1 alpha; CAG: chicken β actin hybrid promoter; PGK: mouse phosphoglycerate kinase; UBC: human ubiquitin C. Relative Gene Expression Normalized to sintc ALDOA down-regulation induced by mmu-mir-122 shmimic in mouse NIH/3T3 cells hcmv sintc hef1α sintc mcmv sintc mef1α sintc mir-122-aldoa Promoters vary in activity in different cells which effects lentiviral microrna expression and, consequently, down-regulation of target gene expression. Mouse NIH/3T3 cells were transduced with SMARTchoice shmimic Lentiviral microrna particles expressing mmu-mir-122, which regulates ALDOA, and a non-targeting control (sintc). Transduction Medium contained 3 µg/ml Polybrene with no serum; cells were incubated overnight with lentiviral particles at MOIs 3, 15 and 7.5. After 72 hours post-transduction, TurboGFP fluorescent intensity was observed by microscopy indicating promoter activity; the mouse CMV (mcmv) promoter is most active in mouse NIH/3T3 cells. Additionally, expression of ALDOA and GAPDH (reference gene) was measured by RT-qPCR using Thermo Scientific TM Maxima TM cdna Synthesis Kit and Thermo Scientific TM Solaris TM Assays and Master Mix. Relative expression of ALDOA was normalized to GAPDH, and then to sintc-treated cells using a ΔΔCq method. Expression of mir-122 by the mcmv promoter resulted in the greatest down-regulation of ALDOA. A Relative ZEB1 mrna Expression Normalized to Matched Negative Control MOI 2 MOI 1 shmimic mir-429 MOI 5 MOI 2 MOI 1 Negative Control MOI 5 MOI 2 MOI 1 Untreated MOI 5 Silencing of ZEB1 expression in MDA-MB-231 cells transduced with mir-429 shmimic microrna. MDA-MB-231 cells were transduced at multiple MOIs with either mir-429 shmimic microrna or negative control. Transductions were performed in triplicate with no puromycin selection. (A) Cells were harvested 12 hours posttransduction and silencing of ZEB1 expression was measured using Verso 2-Step RT-qPCR SYBR Green Kit. Data was normalized to a reference gene (PPIB) and further normalized to the matched shmimic microrna negative controls. (B) Cells were fixed in 2% paraformaldehyde 96 hours post-transfection. Immunofluorescence was used to visualize E-cadherin expression and cellular localization. E-cadherin protein localization is in red, while nuclei stained with Hoechst are in blue. Note the increased expression of E-cadherin in cells transduced with mir-429 shmimic microrna (MOI = 2) as well as the localization of E-cadherin to newly formed adherens junctions. B Untreated mir-429, field 1 Negative Control mir-429, field 2 42 microrna Modulation Technologies microrna Modulation Technologies 43

24 microrna Modulation Technologies I.4 miridian microrna Mimics Individual microrna Libraries: genome-wide and Cherry-pick Controls: negative, positive and transfection Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 microrna Mimic Controls See p. 57 Normalized Luciferase Expression Catalog No.* miridian Mimics and Hairpin Inhibitors Mimic; 2**, 5, 1, C-HMR-XX 2 nmol Hairpin Inhibitor; IH-HMR-XX 2**, 5, 1, 2 nmol * These are agnostic product identifiers. Actual catalog numbers are gene and species-specific (e.g., C ) ** Minimum purchase of four individuals required for 2 nmol quantities miridian microrna Mimic and Hairpin Libraries See p. 74 mir-342 mir-375 mir-21 mir-17 HeLa miridian microrna Mimics are double-stranded RNA oligonucleotides, chemically enhanced with a proprietary design and available for all human, mouse and rat micrornas in the mirbase Sequence Database. Supplement microrna activity with miridian microrna Mimics to study gain-of-function effects Superior performance in comparison to unmodified double-stranded RNA Effective mimic of endogenous mature microrna function to elucidate involvement in biological pathways, identify and validate microrna targets, or screen for micrornas that regulate gene expression Preferential programming of RISC with active strand of microrna and exclusion of passenger strand through a proprietary chemical modification pattern (ON-TARGET) Functional data ALDOA mrna Expression Normalized to Mimic Control #1 (%) mir mir-224 HepG2 mir-185 HUh-7 Control HeLa mir-22 MCF7 mir-32 HEK293 let-7c HepG2 NIH/3T3 mir-18a H9c2 Control miridian microrna Mimic Endogenous Control leads to regulation of Aldolase A in human, mouse and rat cell lines. Many cells lines express low to moderate levels of mir-122. ALDOA is a predicted target of mir-122 and the 3 UTR is conserved in human, mouse and rat at the 8 mer mir-122 predicted seed site. miridian microrna Mimic designed to modulate endogenous mir-122 was transfected at 5 nm (Huh-7 at 4 nm) using DharmaFECT 1 into the indicated cell lines and assessed for their ability to decrease ALDOA mrna levels. ALDOA down-regulation was determined using the branched DNA assay (Panomics Quantigene Reagent System) at 3 days (HepG2 cells at 5 days) post-transfection. miridian microrna Mimics effectively simulate microrna function. miridian microrna Mimic function for 11 human micrornas were assayed in HeLa and HepG2 cells 48 hours after transfection of 1 nm mimic using a dual luciferase reporter system and normalized to the control (no mimic). Similar results were obtained with 1 nm miridian microrna Mimic (data not shown). miridian microrna Hairpin Inhibitors 1 1 miridian microrna Hairpin Inhibitors are single-stranded, chemically enhanced oligonucleotides available for all human, mouse and rat micrornas in the mirbase Sequence Database. This novel microrna inhibitor design is based on findings published by Vermeulen et al. 1 that demonstrate high potency. Innovative design provides superior potency and long-lasting inhibition to study loss-of-function effects Patent-pending molecule combines chemical modifications and novel secondary structure motif Superior potency and longevity in comparison to any other commercially available synthetic microrna product Enhanced potency and longevity allows for multiplexed microrna inhibition at very low nanomolar concentrations with minimal toxicity 2 nm of mir-16 hairpin inhibitor is sufficient to bind or sequester a majority of endogenous mir-16. Endogenous levels of mir-16 were assayed 48 hours after transfection of either miridian mir-16 Hairpin Inhibitor, mir-21 Hairpin Inhibitor or Hairpin Inhibitor Negative Control #1. HeLa cells were transfected using DharmaFECT 1. Detection of mir-16 by RT-qPCR was performed using a poly-a tailing method 2 and Thermo Scientific TM Verso TM 2-Step RT-qPCR SYBR Green Kit according to the manufacturer's protocol. Relative expression of mir-16 was first normalized by mir-21 expression and then further normalized to miridian Hairpin Inhibitor Negative Control #1 levels. Normalized Rluc/Fluc Ratio Functional data Double-stranded region mir-17-5p mir-18a-5p Reverse complement mir-19a mir-2a Double-stranded region mir-19b-1 mir ,6 141,4 121,2 11, Threshold No RT No Template Cycles Fluorescence (dr) Fluorescence (dr) } Relative Expression of mir-16 normalized to Hairpin Control #1 Hairpin mir-16, 5 nm.6% Hairpin mir-16, 2 nm.52% Hairpin Control #1, 5 nm 1% Lipid only 95% Untreated 118% miridian Hairpin Inhibitor Reporter alone Cycles Hairpin mir-16, 2 nm Hairpin mir-16, 5 nm Hairpin mir-21, 2 nm Hairpin mir-21, 5 nm Hairpin Control #1, 2 nm Hairpin Control #1, 5 nm Lipid only Untreated miridian microrna Hairpin Inhibitors allow combinatorial inhibition of clustered micrornas. Six co-expressed micrornas (mir-17-5p, mir-18a-5p, mir-19a, mir-2, mir-19b-1 and mir-92-1) from the Cancer Cluster were inhibited by miridian Hairpin Inhibitors. All six miridian Hairpin Inhibitors were pooled together for a.8 nm total inhibitor concentration. Pools were co-transfected with DharmaFECT Duo into HeLa cells with one of six reporter plasmids specific for each microrna. Data were normalized to similarly treated empty reporter plasmid only. References 1. Vermeulen, A. et al. (27) Double-stranded regions are essential design components of potent inhibitors of RISC function. RNA 13: Shi, R. and Chiang, V.L. (25) Facile means for quantifying microrna expression by real-time PCR. Biotechniques 39: Individual microrna Libraries: genome-wide and Cherry-pick Controls: negative, positive and transfection Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 microrna Hairpin Inhibitor Controls See p. 57 miridian microrna Mimic and Hairpin Inhibitor Libraries See p. 74 I.4 microrna Modulation Technologies 44 microrna Modulation Technologies microrna Modulation Technologies 45

25 Custom Oligonucleotide Synthesis 1.5 Custom Oligonucleotide Synthesis Trust the experts in RNA synthesis chemistry to provide high-quality, single-stranded RNA, DNA, and microrna with an extensive range of modifications, synthesis scales and purification choices. Go beyond pre-designed with custom synthesis Start with a published or a previously purchased catalog item Design your own with the sidesign Center Get expert design services with a custom SMARTpool I.5 Custom Oligonucleotide Synthesis Design your own to target any gene in any species Customize a microrna reagent with chemical modifications Do you want to add modifications to your? Learn more about our custom SMARTpool services at gelifesciences.com/ dharmacon No page 49 page 51 Yes Proprietary modifications Standard modifications Single-stranded RNA synthesis lets you go beyond RNAi. See more application ideas on page 52 Optimize your with proprietary modification patterns page 48 Explore RNAi in vivo with stabilized and special processing page 5 Do you require more than standard purification and processing No Yes Add ON-TARGET or ON-TARGETplus modifications for high specificity Purification typically not required for in vitro use due to high purity from standard processing Add Accell modifications for delivery into cells without a transfection reagent Add HPLC purification for high-sensitivity assays or highly modified s Add sistable modifications for nuclease resistance In vivo processing includes sodium ion exchange and endotoxin testing Add dye, labels, linkers, thiol, biotin, cholesterol and more* Modify the 3' or 5' end of either strand Section I Chapter 5: Table of Contents Custom synthesis selection guide Custom synthesis sidesign Center reagents for in vivo applications... 5 Custom microrna modulation tools Custom RNA synthesis selection guide Custom single-stranded RNA synthesis What quantity of is suitable for your needs? Order online for 2-15 nmol (1 mg) quantities** Suitable for in vivo RNAi * For a comprehensive list of chemical modifications, go to gelifesciences.com/dharmacon ** Larger quantities available upon request. Please contact our Technical Support team. 46 Custom Oligonucleotide Synthesis Custom Oligonucleotide Synthesis 47

26 Custom Oligonucleotide Synthesis 1.5 Custom Synthesis To order custom or learn more about standard modifications available for custom synthesis, go to gelifesciences.com/dharmacon/ rnai-and-custom-rna-synthesis/ *Yields reported should be considered estimates and are not guaranteed amounts. If you have minimum yield specifications for your experiments, please contact our Technical Support Scientists for recommendations regarding synthesis scale. Dharmacon 2'-ACE chemistry is a proprietary RNA synthesis chemistry used in the synthesis of all and microrna products. This synthesis chemistry produces higher yield and purity of the final product compared to other chemistries and is amenable to a broader portfolio of chemical modifications for greater experimental flexibility. Routine production of 8-85% pure unmodified RNA strands without costly purification steps Complete range of customization options include standard chemical modifications such as fluorescent dyes or other molecules, as well as modified nucleobases and linkages Proprietary modifications available for improved specificity, stability and delivery sidesign Center The sidesign Center is a free online design tool that offers the power of the Dharmacon SMARTselection algorithm* for the custom design of highly functional s. User-friendly design tool significantly improves the chances of success for identifying functional s Design an to target single gene variants or cross-species homologs Leverage the power of the SMARTselection algorithm to design s that meet your specific needs To design your sequences using sidesign Center, go to gelifesciences.com/dharmacon/ design-center/ Would you rather have our bioinformatics experts determine your designs? Place your order for Custom SMARTpool production at gelifesciences.com/ dharmacon/rnai-and-custom-rnasynthesis. I.5 Custom Oligonucleotide Synthesis Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 RNAi Controls See p. 54 Product details Place a convenient online order for up to the 1 nmol scale. If larger quantities are desired, please contact Technical Support to request a quote. For unmodified, the following approximate yields can be expected.* µmol scale Quantities available online Standard (A4) Purified in vivo in vivo HPLC nmol mg nmol mg nmol mg nmol mg Basic options Advanced options Design s to target non-standard species or novel genes. Identify the target mrna by providing RefSeq identifiers or FASTA sequence. Design Center :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Design s that target specific gene variants or isoforms. All known RefSeq variants associated with the target gene will be displayed. Select the variant(s) to be targeted by the designs. *Candidates produced using the sidesign Center do not carry the product performance guarantee. However, s designed using this tool are more likely to provide potent and specific knockdown than those designed with conventional methods Step 1: Select an Identifier Type Identifier Type: Enter Accession: Accession Num NM_ Use the radio buttons associated with the Accession number of each variant to Require, Allow or Exclude targeting of candidate designs to its sequence Accession Required Allowed Excluded NM_ , , NM_ ,75 5 1,6 21 2,5 33 1, ,5 1 3,2 42 5, 66 2,5 33 Enter Accession numbers for any required transpript not listed above (e.g., cross-species homolog). Separate multiple entries with a space or a line break. Proprietary modifications These proprietary modifications are available only with Dharmacon pre-designed products or custom synthesis. -specific modifications ON-TARGET ON-TARGETplus Accell sistable Inhibits sense (passenger) strand uptake by RISC Antisense strand seed region modified for greater specificity to target Resistant to endonuclease and exonuclease degradation for enhanced stability Target a specific region of the gene sequence. Select from 5' UTR, ORF or 3' UTR. Cancel Advanced Options ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Step 2: Select desired region(s) for design: 5' UTR ORF 3' UTR ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Step 3: Enter minimum and maximum G/C percentage, or accept the default (recommended) Minimum GC% 3 Maximum GC% 64 ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Step 4: Choose Blast options No BLAST BLAST Database ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: Step 5: Click Design to generate a list of candidate sequences. Design s Design cross targeting to silence homologs in other species or conserved regions of gene family members. Supply up to 1 additional Accession numbers. Delivery into cells without a transfection reagent 48 Custom Oligonucleotide Synthesis Custom Oligonucleotide Synthesis 49

27 Custom Oligonucleotide Synthesis 1.5 Reagents for in vivo Applications To order custom or review citations using sistable or Accell modified for in vivo applications, go to gelifesciences.com/dharmacon/ rnai-and-custom-rna-synthesis/. You may add the sistable or Accell modification pattern to most sequences. Controls: positive and non-targeting Catalog No.* sistable Cyclophilin B control, Mouse 5 nmol and 2 nmol D Specialized offerings address the requirements of a dynamic in vivo environment. In vivo processing, stabilizing modifications and larger quantities are available to enable your RNAi animal studies. Larger synthesis scales provide sufficient material for animal dosing experiments Specialized processing options provide quality suitable for use in mammalian models The proprietary sistable modification pattern dramatically enhances stability in nuclease-rich environments Accell modifications provide enhanced nuclease stability and delivery-enhancing properties, with proven performance in in vivo environments Pre-designed miridian microrna Mimics and Hairpin Inhibitors are available for all human, mouse and rat micrornas annotated in the mirbase microrna database. Customize a pre-designed product, create a mimic or inhibitor for a microrna from an alternative species or design a modulation tool for a novel microrna. Receive custom miridian Mimics, miridian Hairpin Inhibitors or made-to-order microrna reagents Incorporate chemical modifications, introduce mismatches to study microrna biomechanics or design an in vivo experiment Order a miridian stabilized microrna Mimic to enhance nuclease resistance in vivo Order an Accell microrna Inhibitor for delivery into cells without a transfection reagent Custom microrna Modulation Tools To order custom microrna modulation tools, request a quote from our Technical Support Scientists. *A comprehensive list of chemical modifications can be found at gelifesciences.com/dharmacon/rnai-andcustom-rna-synthesis/ Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 I.5 Custom Oligonucleotide Synthesis sistable GAPD control, Mouse 5 nmol and 2 nmol D sistable Non-targeting 5 nmol and 2 nmol D-17-1 * Standard does not require additional processing for in vitro use. **Results typical for unmodified RNA strands. Intact (%) processing options Stability of targeting human cyclophilin B in 1% human serum. The sistable modification pattern dramatically extends the half-life of in the presence of 1% human serum. Unmodified degrades significantly while sistable maintains integrity for up to 5 days. sistable Unmodified Time (hours) Standard (A4)* PAGE (C) HPLC in vivo in vivo HPLC Desalted, deprotected and annealed Purified (9-95%)** Endotoxin tested Sodium counter-ion exchange Recommended for modified (dyes etc.) Recommended for in vivo use A selection of publications demonstrating Dharmacon for in vivo applications: 1. R.H.E. Chong, E. Gonzalez-Gonzalez, et al. Gene silencing following delivery to skin via coated steel microneedles: In vitro and in vivo proof-of-concept. Journal of Controlled Release 166(3), (213). Accell delivered by microneedle treatment to paws (mouse) 2. C. Cheng, R. Haasdijk, et al. Endothelial cell specific FGD5 involvement in vascular pruning defines neovessel fate in mice. Circulation 125(25), (212). Accell delivered by intravitreal injections into the eye (mouse) 3. H. Nakajima, T. Kubo, et al. A rapid, targeted, neuron-selective, in vivo knockdown following a single intracerebroventricular injection of a novel chemically modified in the adult rat brain. J. Biotechnol. 157(2), (211). Accell delivered by injection into the cortical region of the brain (rat) 4. P.A. Singleton, T. Mirzapoiazova, et al. High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness. Am. J. Physiol. Lung. Cell Mol. Physiol. 299, L639-L651 (21). sistable delivered by intrajugular ACE antibodyconjugated liposomal delivery (mouse) 5. M. Snapyan, M. Lemasson, et al. Vasculature guides migrating neuronal precursors in the adult mammalian forebrain via brainderived neurotrophic factor signaling. J. Neuroscience 29(13), (29). sistable using 7s osmotic pump into carotid artery (mouse) 6. H. Watanabe, H. Saito, et al. Activation of phosphatidylinositol-3 kinase regulates pancreatic duodenal Homeobox-1 in duct cells during pancreatic regeneration. Pancreas 36(2): (28). sistable delivered by hydrodynamic tail vein injection (mouse) 7. S.D. Larson, L.N. Jackson, et al. Effectiveness of uptake in target tissues by various delivery methods. Surgery 142(2), (27). Fluorescent-labeled ; a comparison of delivery by hydrodynamic IV injection, standard IV injection, intraperitoneal administration and rectal administration (mouse) 8. N.C. Henderson, A.C. Mackinson, et al. Galectin-3 regulates myofibroblast activation and hepatic fibrosis. Proc. Natl. Acad. Sci. U S A 13(13), (26). sistable delivered by hydrodynamic tail vein injection (mouse) Adapt a catalog miridian Mimic or Hairpin Inhibitor Do you want to add modifications* to your mimic or inhibitor? No Do you require additional purification and processing? Design a mimic or inhibitor to other species in mirbase Yes Yes Yes Design a mimic or inhibitor to a novel microrna Proprietary modifications Order nuclease-resistant Stabilized microrna Mimics Common modifications Add dyes, linkers, thiol, biotin, cholesterol and more* Add HPLC purification for high-sensitivity assays or modified mimics and inhibitors Order selfdelivering, novel Accell microrna Inhibitors Modify the 3' or 5' end of a microrna mimic or inhibitor In vivo processing includes sodium ion exchange and endotoxin testing RNAi Controls See p. 54 Request a quote at gelifesciences.com/ dharmacon/rnai-andcustom-rna-synthesis 5 Custom Oligonucleotide Synthesis Custom Oligonucleotide Synthesis 51

28 Custom Oligonucleotide Synthesis 1.5 Discover new possibilities with customized RNA/DNA oligonucleotides Length Nucleosides Modification position Modification type Linkages Processing Purification Scale Amount Documentation available RNA oligonucleotides support broad applications: microrna functional studies X-ray crystallography, NMR RNA Interference (RNAi) RNA protein interactions Transcription / translation studies Virology RNA biochemistry Common uses for oligonucleotides: Custom microrna inhibitors Aptamers Ribozymes Transfer RNAs (trnas) FISH/ISH or FRET probes RNA/DNA chimeras Custom RNA duplexes Dharmacon offers a broad portfolio of dye modifications for fluorescent labeling of your custom and RNA oligonucleotides. Fluorophore Up to 12 nucleotides (sequence and scale dependent) RNA, DNA, 2'O-Methyl, 2' Fluoro, chimeras, degenerate bases 3' end, 5' end, internal, multiple, sugar or base Fluorescent dyes, quenchers, linkers, thiol, biotin, cholesterol, and more Phosphorothioate Desalting, 2'-ACE deprotection, in vivo HPLC, PAGE, in vivo HPLC.5 µmol to 2 mmol (not all scales available for all oligos) nanomole to millimole (nmol-mmol), microgram to gram (µg-g) Mass spectrometry analysis, UPLC or CGE quality analysis, Certificate of Analysis λ max abs (nm) λ max em (nm) Comparable to 5', 3', internal Custom Single-stranded RNA Synthesis Dharmacon proprietary 2'-ACE RNA synthesis chemistry enables reliable synthesis of RNA with exceptionally efficient step-wise coupling of RNA bases. Short deprotection times and a mild chemical environment promote the highest level of purity for synthetic RNA in the marketplace today. Greater reproducibility with unmatched levels of full-length product Unparalleled yields from other vendors equivalent synthesis scales Unique chemistry capabilities include the synthesis of long RNA oligos and custom chemistry development for novel nucleosides or ACE-adapted phosphoramidites Functional data Sequence fidelity and high yields are the hallmarks of 2' ACE RNA synthesis. For unmodified or shorter RNA oligos (<36 nucleotides), purification is typically not required for in vitro applications due to the high quality of the initial product synthesized using 2' ACE chemistry. This highefficiency chemistry also enables synthesis of highly modified or long oligonucleotides** beyond that of other synthesis platforms. A Absorbance Units (AU) To learn more about our capabilities for oligonucleotide synthesis or to determine feasibility for your project, contact Technical Support. ** Synthesis of long oligonucleotides is sequence and scale dependent. Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 RNAi Controls See p. 54 Long RNA Oligos Unparalleled synthesis capabilities of RNA oligos up to 12 nucleotides. I.5 Custom Oligonucleotide Synthesis Fluorescein / 6-FAM ALL* DY Cy TM 3, Alexa Fluor 546, Alexa Fluor 555 ALL* B Time (minutes) Cy ALL* TAMRA ALL* Cy ALL* DY Cy5, Alexa Fluor 647 ALL* Cy ALL* Cy ALL* DY Cy5.5, Alexa Fluor 68 ALL* Intensity 7, 6, 5, 4, 3, 2, 1, Additional dyes spanning the light spectrum from 35-8 nm are also available upon request. * Ability to fulfill requests for internal dye labels is sequence and length dependent. Mass (Da) Analysis of a 15 nucleotide RNA oligo produced with 2' ACE RNA synthesis chemistry. (A) Analytical ion pairing reversed phase UPLC trace for a 15 nucleotide RNA oligo (with a 5' DMT) showing > 9% full-length material. Oligo was synthesized using 2' ACE chemistry and purified by HPLC. (B) Mass of the 15 nucleotide RNA oligo was analyzed with the LXQ Mass Spectrometer. Results illustrate the correct mass. 52 Custom Oligonucleotide Synthesis Custom Oligonucleotide Synthesis 53

29 RNAi Controls 1.6 RNAi Controls Which RNAi control is best for you? 1.6 RNAi Controls I.6 Critical to any gene silencing or microrna modulation experiment, RNAi controls enable accurate interpretation for reliable results. sigenome Product Line Endogenous Positive Control Lamin A/C, PPIB, GAPD Exogenous Positive Control Negative Control Non-targeting, RISC-Free ON-TARGETplus PPIB, GAPD Non-targeting Fluorescent Label Control Kits Accell PPIB, GAPD egfp Non-targeting sistable PPIB, GAPD Non-targeting Recommended controls for use in RNAi experiments siglo Lamin A/C, PPIB RISC-Free Control type siglo Transfection Indicators Positive control Non-targeting negative control RISC-Free, Empty Vector or mock transfection Fluorescent Controls Validated RNAi reagents to optimize and monitor efficiency of delivery across experimental replicates, conditions or cell types. An RNAi reagent with a non-targeting sequence to provide a baseline reference for target-specific knockdown and distinguish sequence-specific silencing from nonspecific effects. A specialized control or experimental condition that lacks a fully functional RNAi component to detect non-rnai cellular effects (toxicity or gene expression changes) caused by the transfection or transduction. Fluorescent dye-labeled controls, including novel nuclear-localizing Transfection Indicators permit correlation of signal intensity with optimal /microrna transfection conditions. shrna Reporter gene controls RLuc, FLuc, GFP, β-gal, GL2 and GL3 Luciferase Lincode controls GAS5 Non-targeting SMARTchoice Lentiviral shrna PPIB, GAPD Luciferase Non-targeting, empty vector SMARTchoice Inducible shrna PPIB, GAPD Non-targeting GIPZ Lentiviral shrna TRIPZ Inducible Lentiviral shrna EG5, GAPD GAPD Non-targeting, empty vector Non-targeting, empty vector TRC Lentiviral shrna egfp Empty vector micro RNA miridian shmimic Lentiviral microrna miridian microrna Mimics mir-122, 3' UTR controls Non-targeting, empty vector Negative control Section I Chapter 6: Table of Contents miridian microrna Hairpin Inhibitors mir-16 Negative control RNAi controls selection guide Controls ON-TARGETplus Controls Accell Controls sigenome Controls sistable Controls siglo Fluorescent Controls Additional Positive Controls Lincode controls shrna Controls SMARTchoice Lentiviral shrna Controls SMARTchoice Inducible Lentiviral shrna Controls GIPZ Lentiviral shrna Controls TRIPZ Inducible Lentiviral shrna Controls TRC Lentiviral shrna Controls microrna Controls SMARTchoice shmimic Lentiviral microrna Controls miridian microrna Mimic Controls miridian microrna Hairpin Inhibitor Controls Control Kits Accell Control Kits A tool set to assess and optimize Accell technology in your cell type, and to ensure effective controls during initial gene silencing experiments with Accell reagents. Each kit contains: Accell GAPD Control (Human, Mouse, or Rat) Accell Cyclophilin B Control (Human, Mouse, or Rat) Accell Non-targeting #1 Choice of Green or Red Accell Non-targeting 5X Buffer Accell Delivery Media sigenome Control Kits A comprehensive array of control reagents for use in any experiment. Each sigenome Controls Basic Kit contains 5 nmol each of: sigenome Lamin A/C Control (Human/Mouse/Rat) sigenome Cyclophilin B Control (Human/Mouse/Rat) sigenome Non-targeting #2 sigenome Non-targeting #5 sigenome RISC-Free Control siglo Green Transfection Indicator sigenome Non-targeting Pool #2 Each sigenome Controls Complete Kit additionally contains: Three additional siglo control s TOX Transfection Control 54 RNAi Controls RNAi Controls 55

30 RNAi Controls 1.6 Controls, shrna and microrna Controls 1.6 RNAi Controls I.6 Product Size/Format Catalog No. ON-TARGETplus Cyclophilin B Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) ON-TARGETplus Controls ON-TARGETplus Cyclophilin B Control Pool 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) ON-TARGETplus GAPD Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) ON-TARGETplus GAPD Control Pool 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) ON-TARGETplus Non-targeting #1 5, 2, 5 nmol D XX ON-TARGETplus Non-targeting #2 5, 2, 5 nmol D XX ON-TARGETplus Non-targeting #3 5, 2, 5 nmol D XX Product Size/Format Catalog No. Lincode GAS5 Control 5, 2, 5 nmol D XX (H) Lincode GAS5 Control Pool 5, 2, 5 nmol D XX (H) Lincode Non-targeting #1 5, 2, 5 nmol D XX Lincode Lincode Non-targeting #2 5, 2, 5 nmol D XX controls Lincode Non-targeting #3 5, 2, 5 nmol D XX Lincode Non-targeting #4 5, 2, 5 nmol D XX Lincode Non-targeting Pool 5, 2, 5 nmol D XX Accell Controls ON-TARGETplus Non-targeting #4 5, 2, 5 nmol D XX ON-TARGETplus Non-targeting Pool 5, 2, 5 nmol D XX Accell Cyclophilin B Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) Accell Cyclophilin B Control Pool 5, 2, 5 nmol D XX (H), D XX (M), D XX-(R) Accell GAPD Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) Accell GAPD Control Pool 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) Accell Green Cyclophilin B Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) Accell Red Cyclophilin B Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) Accell egfp Control 5, 2, 5 nmol D XX Accell egfp Control Pool 5, 2, 5 nmol D XX Accell Non-targeting #1 5, 2, 5 nmol D XX Accell Non-targeting #2 5, 2, 5 nmol D XX Accell Non-targeting #3 5, 2, 5 nmol D XX Accell Non-targeting #4 5, 2, 5 nmol D XX Accell Non-targeting Pool 5, 2, 5 nmol D XX Accell Green Non-targeting 5, 2, 5 nmol D XX Accell Red Non-targeting 5, 2, 5 nmol D XX Accell Control Kits (Green) Accell Control Kits (Red) 5 nmol each of four controls + delivery media 5 nmol each of four controls + delivery media K-5-G1-1 (H), K-5-G1-2 (M), K-5-G1-3 (R) K-5-R1-1 (H), K-5-R1-1 (M), K-5-R1-1 (R) SMARTchoice Lentiviral shrna Controls SMARTchoice Inducible Lentiviral shrna Controls GIPZ Lentiviral shrna Controls TRIPZ Inducible Lentiviral shrna Controls SMARTchoice Promoter Selection Plate - Viral particles 96-well plate (18 TU/mL) SP-1-1 SMARTvector Empty Vector Control (hcmv-turbogfp) - Viral particles 5 µl 18 TU/mL S-4-1 SMARTvector Firefly Luciferase Positive Control (hcmv-turbogfp) - Viral particles 5 µl 18 TU/mL S-6-1 SMARTchoice Non-targeting shrna Control - Viral particles 5 µl 18 TU/mL S* (H, M, R) SMARTchoice GAPDH shrna Positive Control - Viral particles 5 µl 18 TU/mL S* (H, M, R) SMARTchoice PPIB shrna Positive Control - Viral particles 5 µl 18 TU/mL VS* (H, M, R) SMARTchoice Inducible Non-targeting Control 4-Pack- Viral particles 5 μl 17 TU/mL VSC6847 SMARTchoice Inducible Non-targeting Control - Viral particles 5 μl 17 TU/mL VSC658, VSC6581 SMARTchoice Inducible GAPDH Positive Control - Viral particles 5 μl 17 TU/mL VS* (H,M,R) SMARTchoice Inducible PPIB Positive Control - Viral particles 5 μl 17 TU/mL VS* (H,M,R) pgipz Lentiviral Empty Vector Glycerol stock RHS4349 (H, M) GAPDH-GIPZ Lentiviral shrna Positive Control Glycerol stock RHS4371 (H, M) GAPDH-GIPZ Lentiviral shrna Positive Control - Viral particles 5 μl 1 8 TU/mL RHS4372 (H, M) EG5-GIPZ Lentiviral shrna Positive Control Glycerol stock RHS448 (H, M) EG5-GIPZ Lentiviral shrna Positive Control - Viral particles 5 μl 1 8 TU/mL RHS4584 (H, M) Non-targeting-GIPZ Lentiviral shrna Control Glycerol stock RHS4346 (H, M) Non-targeting-GIPZ Lentiviral shrna Control - Viral particles 5 μl 1 8 TU/mL RHS4348 (H, M) GAPDH-TRIPZ Inducible Lentiviral shrna Positive Control Glycerol stock RHS4744 (H) Non-targeting-TRIPZ Inducible Lentiviral shrna Control Glycerol stock RHS4743 (H) Non-targeting-TRIPZ Inducible Lentiviral shrna Control - Viral particles 5 μl 1 8 TU/mL RHS4827 (H) sigenome Cyclophilin B Control 5, 2, 5 nmol D XX (H, M, R) sigenome GAPD Control 5, 2, 5 nmol D XX (H) sigenome Lamin A/C Control 5, 2, 5 nmol D-15-1-XX (H, M, R) sigenome Non-targeting #1 5, 2, 5 nmol D XX (H, M, R) sigenome Non-targeting #2 5, 2, 5 nmol D XX (H, M, R) sigenome Non-targeting #3 5, 2, 5 nmol D XX (H, M, R) sigenome Non-targeting #4 5, 2, 5 nmol D XX (H, M, R) sigenome Non-targeting #5 5, 2, 5 nmol D XX (H, M, R) sigenome Non-targeting Pool #1 5, 2, 5 nmol D XX (H, M, R) sigenome Non-targeting Pool #2 5, 2, 5 nmol D XX (H, M, R) sigenome RISC-Free 5, 2, 5 nmol D (H, M, R) sigenome Controls Basic Kit 5 nmol each of 7 controls K-28-C4-1 (H, M, R) sigenome Controls Complete Kit (Lamin A/C) 5 nmol each of 11 controls K-28-C2-1 (H, M, R) sigenome Controls Complete Kit (Lamin A/C) 5 nmol each of 11 controls K-28-C2-2 (H), K-28-C2-3 (M), K-28-C2-3 (R) TRC Lentiviral shrna Controls plko.1 Lentiviral Empty Vector Control Glycerol stock RHS48 (H, M) egfp-trc Lentiviral shrna Positive Control Glycerol stock RHS4459 (H, M) sigenome Controls miridian microrna Mimic Controls miridian microrna Mimic Housekeeping Positive Control #1 (PPIB) 5, 2, 5 nmol CP-1-1-XX (H), CP-2-1-XX (M), CP-3-1-XX (R) miridian microrna Mimic Housekeeping Positive Control #2 (GAPDH) 5, 2, 5 nmol CP-1-2-XX (H), CP-2-2-XX (M), CP-3-2-XX (R) miridian microrna Mimic Endogenous Positive Control 5, 2, 5 nmol CP-4-1-XX (H, M, R) miridian microrna Mimic Negative Control #1 5, 2, 5 nmol CN-1-1-XX (H, M, R) miridian microrna Mimic Negative Control #2 5, 2, 5 nmol CN-2-1-XX (H, M, R) miridian microrna Mimic Transfection Control with Dy547 5, 2, 5 nmol CP-45-1-XX (H, M, R) sistable Controls sistable Cyclophilin B Control 5, 2, 5 nmol D XX (H), D XX (M) sistable GAPD 5, 2, 5 nmol D XX (M) sistable Non-targeting #1 5, 2, 5 nmol D-17-1-XX (H, M, R) miridian microrna Hairpin Inhibitor Controls miridian microrna Hairpin Inhibitor Positive Control 5, 2, 5 nmol IP-4-1-XX (H, M, R) miridian microrna Hairpin Inhibitor Negative Control #1 5, 2, 5 nmol IN-15-1-XX (H, M, R) miridian microrna Hairpin Inhibitor Negative Control #2 5, 2, 5 nmol IN-25-1-XX (H, M, R) miridian microrna Hairpin Inhibitor Transfection Control with Dy547 5, 2, 5 nmol IP-45-1-XX (H, M, R) siglo Fluorescent Controls Additional positive Controls siglo Cyclophilin B Control 5, 2, 5 nmol D XX (H, M, R) siglo Lamin A/C Control 5, 2, 5 nmol D XX (H), D XX (M), D XX (R) siglo RISC-Free Control 5, 2, 5 nmol D-16-1-XX (H, M, R) siglo Green Transfection Indicator 5, 2, 5 nmol D XX (H, M, R) siglo Red Transfection Indicator 5, 2, 5 nmol D XX (H, M, R) Anti-Luc 1 2, 5 nmol D-25-1-XX anti β-gal 1 2, 5 nmol D-2-1-XX GFP Duplex 1 2, 5 nmol P XX Luciferase Duplex 2, 5 nmol P XX Luciferase GL2 Duplex 2, 5 nmol D-11-1-XX Luciferase GL3 Duplex 2, 5 nmol D-14-1-XX RLuc Duplex 2, 5 nmol P-27-1-XX siglo Lamin A/C 5, 2, 5 nmol D XX TOX Transfection Control 5, 2, 5 nmol D-15-1-XX XX = 5, 5 nmol; 2, 2 nmol; 5, 5 nmol siglo transfection indicators are the ideal controls to assess efficient uptake in every RNAi experiment for high confidence results. *These are generic product identifiers. Actual catalog numbers will be specific to gene, species, reporter, and promoter. siglo Green Transfection Indicator localizes to the nucleus to confirm successful uptake. Panel (A) illustrates cells successfully transfected with siglo Green that are readily distinguished from non-transfected cells. Panel (B) is the same field of cells stained with a nuclear specific dye (blue nuclei; Hoechst 33342). HeLa cells transfected with siglo Green (2 nm) complexed with DharmaFECT 1 Transfection Reagent (.1 μl/well), fixed with 4% paraformaldehyde and stained with phalloidin- Alexa Fluor- 546 conjugate (Life Technologies) according to manufacturer s protocol. 56 RNAi Controls RNAi Controls 57

31 Delivery Solutions for RNAi 1.7 Delivery Solutions for RNAi Transfection reagents and viral packaging kits for high-efficiency delivery with low cellular toxicity. Which RNAi delivery method is best for you? DharmaFECT Transfection Reagents DharmaFECT Duo Transfection Reagent TurboFECT Transfection Reagent Trans-Lentiviral shrna Packaging Kits 1.7 Delivery Solutions for RNAi Deliver and microrna reagents Co-transfect or microrna with plasmid Achieve efficient and reliable and microrna transfection at low concentrations Package shrna constructs into lentiviral particles with Trans-Lentiviral Packaging Kits Deliver shrna plasmid Package shrna constructs into lentiviral particles Learn more on page page 6 page 63 Transfection reagent ordering guidelines Effectively transfect shrna in a variety of cell types in the presence or absence of serum PLATE TYPE Quantity DharmaFECT Transfection Reagents Approximate number of transfections.2 ml 2-1, TurboFect Transfection Reagent Quantity Approximate number of transfections 96 well.75 ml 75-3,75 1 ml 2,5 page well 1.5 ml 1,5-7,5.2 ml 2.75 ml 75 1 ml ml 1,5.2 ml 1 12 well.75 ml ml 25 Section I Chapter 7: Table of Contents Delivery method selection guide DharmaFECT Transfection Reagents... 6 TurboFect Transfection Reagent Trans-Lentiviral shrna Packaging Kit Ancillary Reagents Stability testing of DharmaFECT transfection reagents well 1.5 ml 75.2 ml 5.75 ml ml ml Delivery Solutions for RNAi Delivery Solutions for RNAi 59

32 Delivery Solutions for RNAi 1.7 DharmaFECT Transfection Reagents Catalog No.* DharmaFECT 1 Transfection Reagents.2 ml T ml T ml T ml T ml T-21-7A DharmaFECT 2 Transfection Reagents.2 ml T ml T ml T ml T ml T-22-7A DharmaFECT 3 Transfection Reagents.2 ml T ml T ml T ml T ml T-23-7A DharmaFECT 4 Transfection Reagents.2 ml T ml T ml T ml T ml T-24-7A DharmaFECT Set of 4 Transfection Reagents 4.2 ml T ml T ml T-25-3 DharmaFECT Duo Transfection Reagents.2 ml T ml T ml T ml T-21-4 DharmaFECT Transfection Reagents are specifically formulated for delivery of small RNAs. Typical results demonstrate higher efficiency with lower cytotoxicity than reagents designed for use with plasmids. While DharmaFECT 1 Reagent is the most all-purpose reagent, DharmaFECT 2, 3 and 4 Reagents offer distinct formulations to provide highly effective delivery of and microrna reagents in a wide variety of cells. Ideal for single-gene studies or high throughput screens Effective delivery for target silencing at low concentrations Low toxicity even at a broad range of experimental conditions for maximum experimental flexibility Co-transfect with a plasmid reporter using DharmaFECT Duo Reagent, the only reagent specifically formulated for the co-delivery of and plasmid DNA Normalized mrna Expression (%) nm 25 nm 5 nm 1 nm 1 nm PPIB MAPK1 PPIB MAPK1 DharmaFECT 1 25 nm 5 nm 1 nm 1 nm 25 nm 5 nm 1 nm 1 nm Competitor Q 25 nm 5 nm 1 nm Cells lines tested with DharmaFECT transfection reagents Choose DharmaFECT 1, 2, 3 or 4 Reagent for optimal transfection of or microrna reagents. The table lists validated transfection recommendations to help you select the appropriate DharmaFECT formulation for your research. Cell line Cell type Recommended DharmaFECT formulation DharmaFECT volume/well (µl) in 96-well plate Plating density in 96-well plate Other successful formulations HUMAN 786-O Kidney adenocarcinoma A549 Lung carcinoma , 3, 4 B PC3 Pancreas adenocarcinoma , 3, 4 DLD-1 Colorectal adenocarcinoma , 3 DU 145 Prostate carcinoma , 3, 4 NCI-H1299 Lung carcinoma HCT-116 Colorectal carcinoma HEK293 Kidney transformed embryonic cells , 4 HeLa Cervical epithelial adenocarcinoma , 3, 4 HeLa S3 Cervical epithelial adenocarcinoma , 2, 3 HepG2 Hepatocellular carcinoma , 2 hmsc Mesenchymal stem cells , 3, 4 HT-29 Colorectal carcinoma , 3, 4 HT-18 Fibrosarcoma , 2, 3 Huh-7 Hepatocarcinoma , 2 HUVEC Umbilical vein endothelial cells , 2 LNCaP Prostate carcinoma MCF-1A Breast adenocarcinoma MCF-7 Breast adenocarcinoma , 4 MDA-MB-453 Breast adenocarcinoma , 3, 4 MDA-MB-231 Breast adenocarcinoma OVCAR-3 Ovarian adenocarcinoma , 3, 4 PC-3 Prostate carcinoma SK-BR3 Breast adenocarcinoma , 3, 4 SK-OV-3 Ovarian adenocarcinoma , 2, 4 U-87 MG Brain glioblastoma , 3, 4 RODENT A7r5 Rat aortic smooth muscle C2C12 Mouse myoblasts , 3, 4 CHO K1 Chinese hamster ovary cells , 2 ES-D3 Mouse embryonic stem cells Visit gelifesciences.com/ dharmacon for additional customer recommendations in over 8 cell lines 1.7 Delivery Solutions for RNAi Optimize delivery with the DharmaFECT Set of 4 (one tube of each formulation) to determine the best transfection reagent for your cell type Working with a new cell type? Check page 61 for recommendations DharmaFECT target mrna levels Competitor Q target mrna levels Cell viability DharmaFECT Reagent provides highly efficient delivery at low concentrations. HeLa cells were transfected with SMARTpool reagents targeting Cyclophilin B (PPIB) or MAPK1 at concentrations of 1, 5, 25, and 1 nm using DharmaFECT 1 and Competitor Q Reagents. mrna expression (bars) was determined by branched DNA assay (Panomics Quantigene Reagent System) and cell viability (data points) was determined by alamarblue Reagent (Biosource International). ES-E14TG2a Mouse embryonic stem cells H9c2 Rat myoblasts , 3, 4 J774A.1 Mouse macrophages NIH/3T3 Mouse embryonic fibroblasts NRK-49F Rat kidney fibroblasts , 4 Rat2 Rat fibroblasts T3-L1 Mouse embryonic fibroblasts OTHER COS-7 African green monkey kidney , 3, 4 All experimental conditions resulted in cell viability and positive control gene silencing of 8% or better. All experiments were performed in 96-well plates with non-targeting control and Cyclophilin B (PPIB) or GAPDH positive control pools at 25 nm; alamarblue Reagent (viability) and knockdown measured at 24 hour post-transfection. Data normalized to untransfected for viability and both untransfected and non-targeting control for gene silencing. Transfection conditions should always be re-evaluated in the context of a new plate format or assay-specific requirements for cell density. 6 Delivery Solutions for RNAi Delivery Solutions for RNAi 61

33 Delivery Solutions for RNAi 1.7 TurboFect Transfection Reagent Catalog No.* TurboFect Transfection Reagent 1 ml sufficient for 3-5 transfections in 96-well format R ml R532 TurboFect Transfection Reagent is a highly efficient, easy-to-use, non-immunogenic transfection reagent. It is a sterile solution of a proprietary cationic polymer in water. The polymer forms compact, stable, positively charged complexes with DNA. These complexes protect DNA from degradation and facilitate gene delivery into eukaryotic cells. Ideal for transfection of a variety of cells, including primary, differentiated and undifferentiated cells Transfection can be performed in the presence or absence of serum Demonstrates superior transfection efficiency and minimal toxicity when compared to lipidbased or other polymer-based transfection reagents Simple 3-step transfection protocol compared with 6-7 steps for other transfection reagents Trans-Lentiviral shrna Packaging Kits Trans-Lentiviral shrna Packaging Kits allows the researcher to generate lentiviral particles containing shrna expression constructs. The resulting viral particles can be used to transduce the cells or tissue of interest for facilitation of robust gene silencing. Effective packaging of second- or third-generation lentiviral vectors Genes encoding the structural components are separated from other genes required for packaging the viral genome to minimize recombination Generate consistent titers of lentiviral particles for the transduction of shrna into dividing and non-dividing mammalian cells ph-stable calcium phosphate transfection reagent has been optimized for achieving the highest viral titers Catalog No.* Trans-Lentiviral shrna Packaging Kit with calcium phosphate 1 reactions TLP reactions TLP reactions TLP5914 Trans-Lentiviral shrna Packaging Kit with calcium phosphate and HEK293T 1 reactions TLP4615 Calcium Phosphate Transfection Reagent 5 reactions TLP5915 HEK293T cell line 1.7 Delivery Solutions for RNAi Trans-Lentiviral shrna Packaging Kit components 1 ml HCL4517 Cells successfully transfected with TurboFect Transfection Reagent Immortalized cell lines B5, BHK21, Calu1, CHO, COLO, COS-7, HEK293, HeLa, HeLa S3, Hep2C, HepG2, Jurkat, L929, RAW264, MDCK, NIH/3T3, Sp2/Ag14, WEHI The packaging system contains all of the necessary components for multiple packaging events and can be ordered with or without the packaging cell line, HEK293T. Alternatively, choose from two bulk packaging systems that contain a larger volume of the packaging mix and transfection reagent sufficient for 5 or 1 packaging events. Primary cell cultures Human lung fibroblasts, rat fibroblasts, mouse bone marrow-derived dendritic cells, mouse bone marrow derived macrophages Component* Function Cell line* HEK293T Allows production of the lentivirus following co-transfection of the transfer plasmid and packaging plasmids in the packaging mix. HeLa Cells 1 2, GFP Positive Cells (%) 1, ,5 1,25 5 1, UT TurboFect Comp A Comp B Mean Fluorescence Intensity WEHI Cells GFP Positive Cells (%) 5 4 1,5 1,25 1, UT TurboFect Comp A Comp B Mean Fluorescence Intensity Packaging mix Transfection reagent* Negative control* * Components that can be ordered separately. Optimized mixture of five packaging plasmids: ptla1- Pak, ptla1-enz, ptla1-env, ptla1-rev and ptla1- TOFF Calcium phosphate GIPZ non-targeting control Supply the helper functions as well as structural and enzymatic proteins in trans required to produce the lentivirus. Optimized ratios of various packaging elements produce maximal titers. Transfection of plasmid DNA into the nucleus of cultured eukaryotic cells. Allows packaging of the expression construct into virions. Viral Proteins HepG2 Cells 6 1,5 GFP Positive Cells (%) 5 1,25 4 1, UT TurboFect Comp A Comp B Mean Fluorescence Intensity Transfection efficiency Mean fluorescence intensity TurboFect Reagent demonstrates higher transfection efficiency and improved plasmid expression across cell lines. Cells were transfected with plasmid DNA encoding enhanced GFP (egfp) with the indicated transfection reagent (UT; untreated control). Transfections were performed according to manufacturers recommendations and subsequent GFP expression was analyzed by flow cytometry. Schematic showing production of lentiviral particles using the Trans- Lentiviral shrna Packaging Kit. Co-transfection of the packaging plasmids and transfer vector into the packaging cell line, HEK293T, allows efficient production of lentiviral supernatant. Virus can then be transduced into a wide range of cell types, including both dividing and non-dividing mammalian cells. Packaging Mix Transfer Vector Transfection Transcription RNA Transduce Cells Titer 1-5 x 1 6 TU/mL Replicationincompetent Virus Assembly & Budding Harvest Viral Supernatant 62 Delivery Solutions for RNAi Delivery Solutions for RNAi 63

34 Delivery Solutions for RNAi 1.7 Ancillary Reagents Catalog No.* 5X Buffer 1 ml B-2-UB-1 Molecular Grade RNase-free Water 1 ml B-3-WB-1 Accell Delivery Media 1 ml B ml B-5-5 DharmaFECT Cell Culture Reagent 1 ml B X Buffer 5X Buffer is appropriate for resuspension and long-term storage of any short, doublestranded or single-stranded synthetic RNA molecule, including modified and unmodified s, microrna Mimics and microrna Hairpin Inhibitors. Dilute with RNase-free water prior to use. amount (nmol) Amount (µl) of 1X buffer to add for desired final stock concentration 1 µm stock 2 µm stock Stability testing of DharmaFECT Transfection Reagents Transfection reagents are often subject to situations in which the shipping and storage conditions fall outside of the recommendations. Shipping delays, accidental placement in a freezer or other circumstances can cause concerns for the product s subsequent performance. In order to simulate these real-life situations, the DharmaFECT transfection reagents were subjected to extremes in temperature and then assessed for delivery efficacy and impact on cell viability. None of the tested conditions are ever recommended for DharmaFECT Reagents, and indeed could have an impact on aspects of transfection not measured here. However, there is a reasonable level of confidence that DharmaFECT products will still be functional following exposure to temperature fluctuation based on the data presented here. 1.7 Delivery Solutions for RNAi , 5 5 Exceeds tube volume Methods DharmaFECT 1, DharmaFECT 2, DharmaFECT 3 and DharmaFECT 4 Reagents were tested at the following conditions to simulate possible shipping/storage conditions as well as a more extreme high temperature (53 ºC). RNase-free Water It is critical to ensure RNase-free conditions to prevent degradation when working with RNA reagents. Molecular Grade RNase-free water is for dilution of 5X buffer or resuspension of RNA. 1. One freeze-thaw cycle at -2 ºC 2. Three freeze-thaw cycles at -2 ºC 3. Room temperature overnight ºC overnight ºC overnight 6. Real shipping conditions: insulated box with an ice pack, left overnight (shipping) 7. UT = untreated Test samples Untreated samples Cell viability Accell Delivery Media Accell requires low-serum or serum-free application conditions. Accell Delivery Media is an enriched, serum-free media specially formulated and optimized for use during Accell delivery to maintain cell viability. A B DharmaFECT Cell Culture Reagent DharmaFECT Cell Culture Reagent (DCCR) is used to dilute DharmaFECT Transfection Reagent prior to use in Reverse Transfection Format (RTF) library plates mrna Level (%) Cell Viability (%) DF 1 DF 2 DF 3 DF 4 DF 1 DF 2 DF 3 DF 4 DF 1 DF 2 DF 3 DF 4 DF 1 DF 2 DF 3 DF 4 DF 1 DF 2 DF 3 DF 4 UT UT UT mrna Level (%) Cell Viability (%) DF 1 DF 2 DF 3 DF 4 DF 1 DF 2 DF 3 DF 4 DF 1 DF 2 DF 3 DF 4 UT UT UT 1 Freeze Cycle 3 Freeze Cycles Room Temp. 4 C Controls 37 C Overnight 53 C Overnight Shipping 4 C Controls DharmaFECT reagents 1, 2, 3 and 4 retained transfection efficiency with low cytotoxicity after being stored at six different sets of conditions. (A) Three conditions were tested: one freeze-thaw cycle at -2 ºC, three freeze-thaw cycles at -2 ºC and room temperature overnight, and compared to the recommended storage conditions of 4 ºC. (B) Three conditions were tested: 37 ºC overnight, 53 ºC overnight and real shipping conditions (insulated box with ice pack left overnight), and compared to the recommended conditions of 4 ºC. For all studies, HeLa cells were transfected with.4 μl DharmaFECT/well and 1 nm human sigenome Cyclophilin B (Catalog #D-466-3) at 1, cells per well. Cell viability was measured with alamarblue, and mrna levels were measured with branched DNA assay (Panomics Quantigene Reagent System) at 24 hours post-transfection. The acceptable cutoff for cell viability is 8%, as denoted by the dotted line. The experimental control samples were untransfected (UT) cells. 64 Delivery Solutions for RNAi Delivery Solutions for RNAi 65

35 RNAi Screening Libraries 1.8 Which RNAi library suits your experimental needs? Ensure RNAi screening success when you choose from, shrna and microrna libraries. Small gene families to genome-wide collections are available in a wide range of screening formats to provide flexible, convenient and high-confidence screening results. Pre-defined Libraries Pre-defined shrna Libraries Reverse Transfection Format (RTF) Libraries Duet Libraries Decode Pooled Lentiviral shrna Libraries miridian Mimic and Hairpin Inhibitor Libraries RNAi Screening Libraries RNAi Screening Libraries Species Available as genome-scale collections Available as custom library Increase hit confidence while reducing the number of hit stratification steps with paired libraries Choose from predesigned or custom RNAi collections for maximum screening flexibility Formats Learn more on page Accomplish multiplexed shrna screening in vitro and in vivo page Follow the icons to your product solution Perform screens without high throughput automation Bacterial glycerol stock: Bacterial culture transformed with a plasmid vector, grown in the presence of glycerol for freezer storage. SMARTpool: A mixture of four s provided as a single reagent. sigenome and ON-TARGETplus SMARTpool reagents guaranteed* to silence by 75% or better. page 7 4 Section I Chapter 8: Table of Contents RNAi Libraries selection guide... Pre-defined Gene Family and Pathway Libraries... Reverse Transfection Format (RTF) Libraries... Duet Libraries... Decode Pooled Lentiviral shrna Libraries... miridian microrna Mimic and Hairpin Inhibitor Libraries... RNAi screening across the workflow... RNAi Screening Libraries page 72 page Set of Four: Four unique s targeting a single gene provided as individual reagents. Three of four sigenome and ON-TARGETplus s are guaranteed* to silence by 75% or better. Pooled high-titer viral particles: Transduction-ready high-titer lentiviral particle format. Gene families and pathways: Pre-defined libraries arranged by gene family and/or function. Arrayed plates: Arrayed in 96-well or 384-well microtiter plates. Species: Human * sigenome and ON-TARGETplus reagents (SMARTpool and at least three of four individual s) are guaranteed to silence target gene expression by at least 75% at the mrna level when used under optimal delivery conditions (confirmed using validated positive and negative control s). Silencing should be monitored at the mrna level hours after transfection using 1 nm. Mouse Rat Need a library that you configure with your own gene list? Find out more about Cherry-pick Libraries on page 27 RNAi Screening Libraries 67

36 RNAi Screening Libraries 1.8 Pre-defined Gene Family and Pathway Libraries Don t see a gene collection of interest in the adjacent tables? Configure a library of your favorite genes with the Cherrypick Library Loader at gelifesciences. com/dharmacon/libraryimport/ For additional information or assistance in placing orders, contact Technical Support. Dharmacon pre-defined libraries have been assembled using innovative bioinformatic tools to reflect the latest genetic and ontological information from peer-reviewed publications and databases, thus providing the most up-to-date libraries for your gene-silencing experiments. Available for popular gene families and pathways with sigenome, ON-TARGETplus and Accell reagents or GIPZ and TRC lentiviral shrna reagents Human and Mouse Whole Genome and shrna libraries have been placed in leading research institutions worldwide Landmark publications demonstrate the contribution of these important research tools to the basic understanding of disease states HUMAN (number of genes) MOUSE (number of genes) Pre-defined Libraries sigenome ON-TARGETplus Accell sigenome ON-TARGETplus Accell Ion Channels Phosphatases Protein Kinases Ubiquitin Conjugation Subset # Ubiquitin Conjugation Subset # Ubiquitin Conjugation Subset # Deubiquitinating Enzymes RNAi Screening Libraries Drug Targets 4,795 4,786 4,799 7,1 5,144 Druggable Genome 7,562 7,553 7,587 1,6 6,457 Ion Channels Phosphatases GPCRs Proteases Ubiquitin Conjugation (Subsets 1, 2, 3) Kinases G Protein-Coupled Receptors (GPCR) Proteases Apoptosis Cell Cycle Regulation Nuclear Receptors Tyrosine Kinases Genome 18,144 18,9 19,98 Drug Targets Membrane Trafficking Cytokine Receptors DNA Damage Response Serine Proteases Transcription Factors 1,529 1,44 Epigenetics Drug Target Subcategories Anti-apoptosis Autophagy Enzymes Transcription Regulators Transporters Transmembrane Receptors GPCR Signaling DNA Repair Translation Regulators Senescense Nuclear Receptors Apoptosis Signaling Cell Cycle HUMAN (number of genes) MOUSE (number of genes) Pre-defined shrna Libraries GIPZ TRC GIPZ TRC Ubiquitin Conjugation (combined subsets) Protein Kinases Phosphatases The Druggable Genome Libraries are organized by relevant gene family subsets. G Protein-Coupled Receptors (GPCR) ,29 Druggable Genome 4,753 7,349 6,8 Ion Channel 348 Proteases 478 Epigenetics Gene number may fluctuate as libraries undergo revision or when gene records are updated. For more gene family libraries and availability, go to gelifesciences.com/ rnai-and-customrna-synthesis/rnaiscreening-libraries RNAi Screening Libraries RNAi Screening Libraries 68 69

37 RNAi Screening Libraries 1.8 Reverse Transfection Format (RTF) Libraries Don t see a gene collection of interest? Submit your gene list for custom RTF library fulfillment (2-well, eight-plate set minimum order) at For additional information or assistance in placing custom library orders, contact Technical Support. Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Cell Culture Reagent (DCCR) See p. 64 DharmaFECT Transfection Reagents See p. 57 Controls See p. 74 Dharmacon Reverse Transfection Format (RTF) SMARTpool Libraries are ideal for the lab that wants to screen with, but doesn t have access to high throughput automation. SMARTpool and validated control reagents are pre-dispensed at assay-ready amounts in replicate 96-well cell culture plates. No special handling is required prior to use. Simply resuspend the and add your cells. Each library is supplied as six replicate plate sets containing 6.25 pmol of per well; sufficient to run two screens in triplicate at 5 nm concentration (additional plate sets available) RTF Optimization Plates are available to optimize transfection, controls and cell density for your assay prior to beginning your screen RTF libraries are available in clear-bottom plates with clear-, black- or white-walled wells to support assays involving fluorescent or luminescent detection methods (cover plates are default option) Step 1 Prepare rehydration solution Step 2 Rehydrate RTF plate (25 µl/well) Predispensed dehydrated DharmaFECT Cell Culture Reagent DharmaFECT Transfection Reagent Optimization Plates for RTF libraries Determining appropriate experimental conditions is a critical component to successful screens. RTF Optimization Plates are designed for optimization of cell density, transfection reagent amount and determination of the best non-targeting control for your assay. Each kit contains: Three ready-to-use plates of validated RNAi controls at 6.25 pmol per well Black or white clear-bottom plates available to support assays involving fluorescent or luminescent detection Nontargeting s Non-targeting pool Positive Control No A B C D E F G H ON-TARGETplus RTF Optimization Plates, catalog numbers of controls included: Human or Mouse A ON-TARGETplus Non-targeting #1 D B ON-TARGETplus Non-targeting #2 D C ON-TARGETplus Non-targeting #3 D D ON-TARGETplus Non-targeting #4 D E ON-TARGETplus Non-targeting pool D F ON-TARGETplus Human or Mouse Cyclophilin B Control Pool D or D sigenome RTF Optimization Plates, catalog numbers of controls included: Human/Mouse A sigenome Non-targeting #2 D B sigenome Non-targeting #3 D C sigenome Non-targeting #4 D D sigenome Non-targeting #5 D E sigenome Non-targeting pool #2 D F sigenome Cyclophilin B (H, M, R) Control D RNAi Screening Libraries Step 3 Add cells in appropriate serum-containing media (1 µl/well) Step 4 Perform functional assay for detectable cellular response Transfection Reagent: complex formation Gene Silencing Screening data courtesy of GE Healthcare, formerly Amersham Biosciences. HUMAN Catalog No. MOUSE Catalog No. RTF Libraries sigenome ON-TARGETplus sigenome ON-TARGETplus Optimization Plates H-22* H-122* H-22* H-1122* Apoptosis H-395 H-139 H-139 H-1139 Cell Cycle Regulation H-325 H-132 H-132 H-1132 Cytokine Receptors H-45 H-14 H-14 Deubiquitinating Enzymes H-475 H-1475 H-147 H-1147 DNA Damage Response H-65 H-165 RTF Optimization Plates are recommended to determine optimal screening conditions in your chosen cell type Simple four-step protocol for screening with RTF SMARTpool Libraries. G Protein-Coupled Receptors (GPCR) H-365 H-1365 H-1365 H-1136 Ion Channels H-385 H-1385 H-1385 H-1138 Reverse Transfection Format is highly effective across cell lines. Eight cell lines were transfected with control s targeting Cyclophilin B using RTF plates. Good cellular viability and effective target silencing were accomplished under optimized conditions. Gene knockdown is normalized to GAPDH expression. Cell plating densities are indicated in parentheses. Gene expression was determined 48 hours post-transfection by branched DNA assay (Panomics Quantigene Reagent System; pink bars). Cell viability was determined by alamarblue metabolic assay (grey circles) Screening 4 data courtesy of GE Healthcare, formerly Amersham Biosciences Target Expression (% of Control) HeLa (1,) Expression Viability DU 145 (1,) A549 (1,) PC-3 (1,) HEK293- Luc (25,) Cell Lines (Cell Density) HepG2 (1,) HeLa S3 (1,) HUASMC (25,) Cell Viability (Relaitve to Control) Membrane Trafficking H-555 H-155 H-155 Nuclear Receptors H-345 H-134 H-134 Phosphatases H-375 H-1375 H-1375 H-1137 Proteases H-515 H-1515 H-515 Protein Kinases H-355 H-1355 H-1355 H-1135 Serine Proteases H-535 H-153 Tyrosine Kinases H-315 H-131 Ubiquitin Conjugation Subset #1 H-5615 H-1561 H H Ubiquitin Conjugation Subset #2 H-5625 H-1562 H H Ubiquitin Conjugation Subset #3 H-5635 H-1563 H H Epigenetics *Catalog number shown is for clear plates. Add suffix B or W to indicate black or white walled plates. H-xx22-D includes 1 ml DCCR. DharmaFECT Cell Culture Reagent (DCCR) is recommended for use with RTF libraries to dilute transfection reagents prior to use ( p. 62). DharmaFECT Transfection Reagents are highly recommended for use with RTF libraries, and should be purchased separately ( p. 58). 7 RNAi Screening Libraries RNAi Screening Libraries 71

38 RNAi Screening Libraries 1.8 Duet Libraries Duet Libraries Catalog No. HUMAN Drug Targets G Druggable Genome G G Protein-Coupled Receptors (GPCR) G-4365 Genome G-455 Ion Channels G-4375 Phosphatases G-4375 Proteases G-4515 Protein Kinases G-4355 Ubiquitin Conjugation Subset #1 Ubiquitin Conjugation Subset #2 Ubiquitin Conjugation Subset #3 Related products 5X Buffer See p. 64 RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 Controls See p. 56 G G G Duet Libraries are paired collections of sigenome and ON-TARGETplus SMARTpool reagents, providing knockdown by the two most reliable reagents available. This unique screening format allows faster identification of high-confidence hits. Move high-quality hits through the pipeline faster by screening with Duet libraries; save on downstream hit stratification by reducing the need for secondary screening Cost-effective and guaranteed collections of highly potent reagents, offer support for multiple assays, cell lines or primary screen follow up Deconvolution of up to eight individual s for high-confidence hit stratification Primary Screen To identify potential gene targets SMARTpool Individual s Duet sigenome & ON-TARGETplus SMARTpool Hit Stratification Phenotype confirmation with multiple reagents Individual s Second SMARTpool Target Validation Multiple assays Additional cell types Phenotype rescue Pathway mapping In silico analysis Duet libraries support high-confidence screening workflows. By leveraging the high-efficiency silencing of market-leading reagents, the execution of a Duet screen with two unique SMARTpool reagents (sigenome and ON-TARGETplus) increases overall primary hit quality and reduces follow-up experimental time and effort. 4 Decode Pooled Lentiviral shrna Libraries Decode Pooled Lentiviral shrna Screening Libraries are pools of GIPZ shrnas targeting human genes, provided as concentrated lentiviral particles. Through RNAi-mediated silencing of hundreds or thousands of genes in parallel, a pooled lentiviral shrna screen can be performed to identify genes that regulate cellular responses and signaling pathways, or to discover novel gene functions. In contrast to the costly automated techniques that are required to screen using individually arrayed RNAi reagents, Decode pooled shrna screening libraries allow the researcher to transduce and screen a population of cells within a few tissue culture dishes. High-quality shrna pools provided as concentrated lentiviral particles Non-targeting control for transduction optimization Optimized and validated primers for efficient PCR amplification of genomic DNA with minimal bias High-throughput multiplexed sequencing for hit identification Experimentally tested protocols and planning tools for successful pooled lentiviral RNAi screens Product Details Library Catalog Number Number of genes targeted Number of pools x number of shrna constructs per pool Lentiviral particle volume per pool Ubiquitin Conjugate RHS pool of 3,83 shrna 2 tubes x 25 µl (5 µl total) Phosphatase RHS pool of 1,561 shrna 2 tubes x 25 µl (5 µl total) Protein Kinase RHS pool of 4,675 shrna 2 tubes x 25 µl (5 µl total) Ion Channel RHS pool of 1,884 shrna 2 tubes x 25 µl (5 µl total) GPCR RHS pool of 2,591 shrna 2 tubes x 25 µl (5 µl total) Protease RHS pool of 2,559 shrna 2 tubes x 25 µl (5 µl total) Druggable Genome RHS pools of 8,49 shrna 4 tubes x 25 µl (1 µl total) Human Genome RHS pools of 9,57 shrna 4 tubes x 25 µl (1 µl total) Assay development and optimization Transduction parameters Screening parameters Selective pressure Pooled shrna screening workflow. Assay Development and Optimization: Establish optimal experimental conditions, including those for a) lentiviral transduction and b) screening parameters, such as selective pressure and time between collection of reference and experimental samples. See list under Product Details. Catalog No. Ancillary reagents Decode Indexing PCR and Sequencing Primer Kit Related products GIPZ shrna Controls See p. 55 PRM6178 GIPZ Lentiviral shrna See p RNAi Screening Libraries Relative Target Expression (%) sigenome ON-TARGETplus 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm 5 nm 1 nm 2 nm PIK4CA PTPRJ TESK1 MA PK3 A KA P11 STK39 A KT2 GSK3A ITGB1BP1 STK17A Target Gene sigenome and ON-TARGETplus SMARTpool reagents are highly potent. Target mrna knockdown in a screen with sigenome and ON-TARGETplus SMARTpool reagents was highly effective, even at concentrations of 2 nm. In extended studies, ON-TARGETplus also was effective in reducing false phenotypes due to off-targets. Primary screen Hit identification Reference sample Log Experimental/Reference Transduce with viral shrna pool Select transduced cells Select phenotype Isolate gdna and analyze sequence of integrated shrna Log mean counts Experimental sample Selective pressure Enriched shrnas Depleted shrnas Primary Screen: A stable population of cells expressing single integrants of shrnas are created by transducing Decode lentiviral pools at low MOIs. Transduced cells are then split into reference and experimental populations for application of a selective pressure that induces the phenotype of interest. Genomic DNA (gdna) is then isolated from reference and experimental populations of transduced cells. Illumina-adapted primers and Illuminaadapted primers and a high fidelity polymerase are used to PCR amplify integrated shrna sequences and add Illumina flow-cell binding sequences. The resulting amplicons are run on Illumina platform sequencers, using the sequencing primers provided. Hit Identification and Follow up: shrna sequences are identified in reference and experimental libraries. shrnas that are enriched or depleted during the screen are identified as hits, and the genes that they target are identified. Hits can be confirmed and studied further using individual shrna constructs that can be ordered from the GIPZ lentiviral shrna collection. References 1. Gazin, C. et al. (27) An elaborate pathway required for Ras mediated epigenetic silencing. Nature 449: Gobeil, S. et al. (28) A genomewide shrna screen identifies GAS1 as a novel melanoma metastasis suppressor gene. Genes and Development 22: Hurov, K.E. et al. (21) A genetic screen identifies the triple T complex required for DNA damage signaling and ATM and ATR stability. Genes and Development 24: Schalbach, M.R. et al. (28) Cancer proliferation gene discovery through functional genomics. Science 319: Silva, J.M. et al. (28) Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319: Westbrook, T.F. et al. (25) A genetic screen for candidate tumor suppressors identifies REST. Cell 121: RNAi Screening Libraries RNAi Screening Libraries 73

39 RNAi Screening Libraries 1.8 miridian microrna Mimic and Hairpin Inhibitor Libraries To order a custom collection of miridian microrna reagents, use the Cherry-pick Library Loader (gelifesciences.com/ dharmacon/libraryimport) to upload your list, configure your plates, and place an order. For additional information or assistance in placing custom library orders, contact Technical Support or your local sales representative. Catalog No.* HUMAN MOUSE RAT miridian microrna Mimic Libraries CS-12 CS-22 CS-32 Molecular Grade RNase-free Water IH-12 IH-22 IH-32 Related products 5X Buffer See p. 64 miridian microrna Mimic and Hairpin Inhibitor libraries are available for every human, mouse and rat microrna in the mirbase Sequence Database. miridian libraries are packaged in 96-well plates for high-throughput phenotypic screening applications. Mimics are modified to prevent sense strand uptake and ensure results are due to the mature microrna Hairpin Inhibitors have long-lasting and potent inhibition, typically allowing enough time for complete phenotypes to develop Functional Data A miridian microrna Hairpin Inhibitor library screen was used to identify micrornas involved in human mesenchymal stem cell (hmsc) osteogenesis. hmsc are multipotent cells that can differentiate into osteoblasts or adipocytes, among others. The inhibition or up-regulation of certain micrornas (or the combined inhibition and up-regulation of multiple micrornas) can drive cell fate by creating a cellular milieu specific for differentiation into one cell type versus another. A Assay development Phenotypic screening Hit identification Hit validation Differentiation assay; robust phenotypic marker Human microrna inhibitor library in triplicate Dose effects Human microrna mimics RNAi screening across the workflow Partner with the RNAi leader for your screening needs. We offer technologically advanced, shrna and microrna reagents for highly efficient genome-wide RNAi. Innovation Our market leadership begins with scientific contributions to the field of RNAi that have facilitated key technological advances in the development of potent and specific, microrna-adapted shrna and microrna reagents. Total workflow solutions Simplify experimental workflows by accessing a broad portfolio of molecular biology tools, including controls, delivery reagents, multiple RNAi reagents for hit stratification and ancillary tools for RNAi rescue and detection. RNAi screening expertise Providing innovative RNAi research tools, we also employ these tools in high throughput library screens in research and development. As such, we provide unparalleled expert guidance for genome-scale RNAi experimentation and infrastructure. Cell Culture Assay Development Automation Optimization High Throughput Screening 1.8 RNAi Screening Libraries RNase-free Water See p. 64 DharmaFECT Transfection Reagents See p. 6 midirian microrna Mimic and Hairpin Inhibitor Controls See p. 57 SMARTchoice shmimic Lentiviral micrornas See p. 42 Z score B MicroRNA target validation Putative targets of microrna; Confirm phenotype with AP activity C Hit Selection Target Validation -2-4 Inhibition of micrornas dramatically affects osteogenic differentiation of hmsc. (A) Schematic showing a typical microrna screening workflow. Alkaline Phosphatase (AP) activity was used as an assay of differentiation. miridian microrna Hairpin Inhibitor library targeting mirbase 8. was used in the primary phenotypic screen. miridian microrna mimics were used in the hit validation. (B) Fifteen microrna inhibitors with Z score values of two standard deviation or more were identified as putative hits in the primary phenotypic screen for their effect on AP activity in differentiated hmsc. Data is representative of a single screen (performed in triplicate). (C) Seven inhibitors were confirmed as hits in separate experiments. The Y axis is a measure of AP activity as a biomarker for osteogenic differentiation. The effect of inhibitor (yellow) on AP activity compared to effect of mimic (blue) is shown. microrna mimics for four of the seven micrornas demonstrated no significant effect on AP activity (p-value >.1): mir-189, -153, -133a and For three other micrornas, mir-148b, -27a and -489, mimics had a significant effect on AP activity (p-value <.1), and was an opposite effect to that of the corresponding inhibitor. The microrna mimic and inhibitor effect on AP activity was normalized to mimic negative control 1 (mc1) and inhibitor negative control 1 (ic1), respectively. Data are representative of three independent experiments performed in triplicate. Figures adapted from Schoolmeesters et al. 29. See published citation for full experimental details. 1 2 mir-489 mir-189 mir-153 mir-27a mir-133a mir-486 mir-148b ic1 mc1 Community The RNAi Global Initiative is a unique alliance of leading international biomedical researchers established to accelerate the utility of genome-scale RNAi screening. Led by Dharmacon, the Initiative provides a forum for scientific collaboration, protocol sharing, establishment of experimental standards and tool development for data analysis with a mission to accelerate biological research and medical discovery. Learn more at References 1. Schoolmeesters, A. et al. (29) Functional profiling reveals critical role for microrna in differentiation of human mesenchymal stem cells. PLoS One 4(5):e RNAi Screening Libraries RNAi Screening Libraries 75

40 Gene Expression II. II. Gene Expression Section II Gene Expression Chapter 1: Introduction to Gene Expression Chapter 2: Pre-made cdnas and ORFs Chapter 3: Non-mammalian Resources Gene Expression 76 Gene Expression 77 77

41 Gene Expression II.1 Gene over-expression as a tool for gene analysis and protein science Over-expressing genes can offer insight into gene function at multiple levels. Gain-of-function experiments often result in phenotypic changes that provide the counterpart to loss-of-function techniques such as RNAi. cdna and Open Reading Frame (ORF) expression vectors provide unique tools for understanding gene function through modulating gene expression, but more specifically, protein function. Depending on the expression vector design, additional peptide sequences or tags can be added to the protein of interest, conferring new properties to the native protein. Affinity tags are used to purify the protein for further functional or structural studies. Epitope tags can be added to standardize detection or purification via antibodies. Fluorescent tags can be used to study cellular localization, and reporters can be used to monitor gene expression. The progression of vector technology along with the growth of large genome-scale cdna and ORF collections has resulted in a number of validated solutions for over-expression experiments that provide additional insight into gene function and regulation. Expression plasmid Translation Nucleus Transcription mrna 5' UTR ORF 3' UTR cdnas are derived from mrna. cdnas contain the ORF as well as all or part of the 5' and 3' untranslated regions (UTRs). att cdna clone ORF clone ORF att ORF clones are created from cdna clones by removing the untranslated regions (UTRs) leaving just the protein coding open reading frame, or ORF. The UTRs can be replaced by recombinational cloning sites (such as att sites) for easy vector transfer. The importance of full insert sequencing cdna clones curated through the Mammalian Gene Collection (MGC) 1 pipeline have been fully sequenced to verify the inclusion of a complete coding sequence (CDS). Following full-insert sequencing, approximately 45% of putative full-length clones in the MGC pipeline were eliminated for various reasons including incomplete ORFs, chimeric sequences or frameshifts. The widespread phenomenon of alternative splicing provides further motivation for full-insert sequencing of cdna clones. It is estimated that 74% of multi-exon genes are alternatively spliced. 2 Thus, clones that are only end-sequenced have a significant risk of being assigned an incorrect reference accession. As an example, multiple transcript variants of the DNA methylase gene (DNMT3B) can be expressed from a single cell line. 3 Despite only small transcript size differences, these variants have dramatic effects on cells. Expression of DNMT3B7 splice variant has been shown to change gene expression profiles and alter genome methylation states of HEK293 cells to mimic those seen in cancer cell types. Without full-insert sequencing, this splice variant would be indistinguishable from the wild-type transcript. De novo costs cdna synthesis and cloning technology are well-established and performed in many labs, but the actual cost in time and money is often over-looked. Creating your own clone requires numerous days and there are several steps in the cloning process that could introduce mutations or fail outright. Additionally, a transcript that is successfully cloned may not be the desired sequence variant. of pre-made clones cdna or ORF clones from existing collections provide an alternative that overcomes the expense and uncertainties of de novo cloning. A simple gene search on the GE Lifesciences website will provide links to cdna and ORF clones. Once a fully sequenced clone is identified, researchers can compare the sequence of the clone with their desired sequence, ensuring a suitable match. Purchasing pre-made cdna or ORF clones is often faster and can be less expensive when considering the time required for cloning can now be dedicated to the experiment. 9 9 Alternative splicing events results in two variant cdnas for the DNMT3B gene. (A) The wild-type transcript produces the full-length protein. (B) The variant occurs when an aberrant splicing event excludes exon 1 and retains intronic sequence. Exons outside of this area are unaffected, and the two isoforms would be indistinguishable via end sequencing and gel electrophoresis. Gene search Fully-sequenced clone delivered 1 11 a 12 Save time and money with pre-made cdna and ORF clones A B 11 b Determine your gene of interest Harvest cells RNA purification cdna synthesis Gene-specific PCR Gel sizing and purification Ligate and transform 12 Screen and fully sequence insert II.1 Gene Expression Phenotype Localization Purification Once in the nucleus, the cellular machinery recognizes the promoter, transcribes the protein coding sequence into mrna and translates the protein. The resulting gene product can cause alterations in phenotype, be purified for further processing or be tracked via localization to better understand protein function. References 1. MGC project team (29) The completion of the Mammalian Gene Collection (MGC). Genome Research 19: Johnson, J.M. et al. (23) Genome-wide survey of human alternative pre-mrna splicing with exon junction microarrays. Science 32: Ostler, K.R. et al. (27) Cancer cells express aberrant DNMT3B transcripts encoding truncated proteins. Oncogene 26(38): Start your experiment in DAYS Start your experiment in WEEKS 78 Gene Expression Gene Expression 79

42 Pre-made cdnas and ORFsII.2 II.2 Pre-made cdnas and ORFs Comprehensive collections of pre-made cdnas and ORFs Which gene expression solution is best for you? cdnas Mammalian Gene Collection (MGC) Collections of pre-made, sequence-verified cdna and open reading frame (ORF) clones for reliable gene over-expression. ORFs CCSB Human ORFeome Human ORFeome V8.1 ORFeome Collaboration CCSB-Broad Lentiviral Expression ORF Library Precision LentiORF Species Fully sequenced Expression-ready Genome-scale cdna and ORF collections Expression-ready clones to accelerate your research subsets Easy-to-transfer Fluorescent reporter gene GFP Ready-to-use page 82 Genome-scale page 86 and 87 Formats Learn more on page number Easy-to-transfer gene content Working with difficult-to-transfect or non-dividing cells? page 85 page 87 Gene Expression Follow the icons to your product solution Bacterial glycerol stock: High-titer viral particles: Bacterial culture transformed with a plasmid vector, supplied in glycerol-containing medium for freezer storage and supplied in single tubes. Transduction-ready high-titer lentiviral particle format. Arrayed library format: Section II Chapter 2: Table of Contents cdna and ORF selection guide... Mammalian Gene Collection... CCSB Human ORFeome Collection... Human ORFeome V ORFeome Collaboration Collection... CCSB-Broad Lentiviral Expression Library... Precision LentiORF Collection... Pre-defined Gene Family cdna & ORF Libraries... TurboFect Transfection Reagent... TurboFect in vivo Transfection Reagent... Trans-Lentiviral ORF Packaging Kit Genome-scale or custom libraries of bacterial glycerol stocks arrayed in 96-well microtiter plates. Precision LentiORF Starter Kits: All reagents and protocols necessary to start a gene expression experiment are provided in a single, convenient package. Species: Gene families and pathways: Pre-defined libraries arranged by gene family and/or function and arrayed in 96-well microtiter plates. GFP Human Mouse Rat Fluorescent markers: Visually track expressed protein expression with TurboGFP (green fluorescent protein). * Derived from fully sequenced ORFeome Collaboration clones. Gene Expression 81

43 Pre-made cdnas and ORFs II.2 Choose your pre-made cdna or ORF Dharmacon gene expression products include comprehensive collections of pre-made cdnas and ORFs. Sequence verified and genome-scale cdnas, Gateway adapted ORFs and expression-ready lentiviral ORFs are available as single clones, custom rearrays or libraries and are guaranteed to match sequences in GenBank. Mammalian Gene Collection (MGC) Setting the standard for gene expression, the Mammalian Gene Collection 1, 2, 3 is a fully sequenced, genome-scale collection of cdnas for human, mouse and rat. Developed by the National Institutes of Health (NIH), it is the most extensive, rigorously sequenced collection of mammalian cdna clones. These high-quality collections have been rearrayed into gene family libraries with expression-ready content. High-quality human, mouse and rat cdna clones Sequence-verified to contain a complete ORF Sequence guaranteed to match Genbank published sequence Pre-made genome-scale collections Individual clone glycerol stock Libraries: pre-defined gene families or custom rearray glycerol stock Genome-scale library glycerol stock Related products Precision LentiORF Clones and Libraries See p. 87 Gene Family Libraries See p. 88 TurboFECT Transfection Reagent See p. 89 II.2 Pre-made cdnas and ORFs MGC cdna clones 3,39 27,457 6,769 Fully sequenced cdna Collections Mammalian Gene Collection (MGC) High-quality human, mouse and rat cdna clones Fully sequenced and verified to contain a complete coding sequence (CDS) as represented in GenBank MGC Non-redundant genes 17,421 17,285 6,363 Number of clones and unique genes covered by the Mammalian Gene Collection. These may fluctate as libraries undergo revision or when gene records are updated. 5' UTR ORF 3' UTR For an MGC cdna to be called "full-length", it must contain the ORF as well as all or part of the 5' and 3' untranslated regions (UTRs). Expression-ready MGC subset Fully sequenced CMV promoter for robust expression CCSB Human ORFeome Collection Gateway-adapted ORF Collections CCSB Human ORFeome Collection Native stop codons removed Broad, genome-wide coverage Human ORFeome V8.1 Fully sequenced Native stop codons removed ORFeome Collaboration Collection Fully sequenced With or without a native stop codon Derived from fully sequenced Mammalian Gene Collection (MGC) cdnas and cloned into a Gateway recombinational entry vector at the Cancer Center for Systems Biology (CCSB) and Dana- Farber Cancer Institute, 4 the Human ORFeome collection is ideal for easily moving thousands of open reading frames into your choice of protein expression systems. For maximum flexibility, the native stop codon has been removed. Gateway-adapted ORF clones Fusion-ready with native stop codons removed Individual clone glycerol stock Libraries: pre-defined gene families or custom rearray glycerol stock Genome-scale library glycerol stock Catalog No. CCSB Human ORFeome Collection CCSB human ORFeome Clone CCSB human ORFeome Collection OHS177 OHS5177 Expression-ready ORF Collections CCSB-Broad Lentiviral Expression Collection Sequence-confirmed expression-ready lentiviral ORFs Clones provided as glycerol stock Precision LentiORFs Expression-ready lentiviral ORFs Bacterial glycerol stocks or purified, high-titer lentiviral particle format References 1. MGC Program Team (22) Generation and initial analysis of more than 15, Full-length Human and Mouse cdna Sequences. PNAS 99(26): MGC Project Team (24) The status, quality, and expansion of the NIH Full-length cdna Project: The Mammalian Gene Collection (MGC) Genome Res. 14: MGC Project Team (29) The completion of the Mammalian Gene Collection (MGC). Genome Res. 19: Lamesch, P. et al. (27) horfeome v3.1: A resource of human open reading frames representing over 1, human genes. Genomics 89: Human ORFeome clones have their native stop codon removed to allow the addition of C-terminal fusion tags Related products Gene Family Libraries See p. 88 TurboFECT Transfection Reagent See p. 89 Pre-made cdnas and ORFs Pre-made cdnas and ORFs 82 83

44 Pre-made cdnas and ORFs II.2 Human ORFeome V8.1 Individual clone glycerol stock Libraries: custom rearray glycerol stock Genome scale library glycerol stock Human ORFeome V8.1 horfeome v8.1 Clone horfeome v8.1 Library horfeome v8.1 Supplemental Library Related products Catalog No. OHS684 OHS686 OHS69 TurboFECT Transfection Reagent See p. 89 CCSB-Broad Lentiviral Expresion ORF Library See p. 86 The Human ORFeome v8.1 represents the most recent version of the Human ORFeome from the Center for Cancer Systems Biology at Dana-Farber Cancer Institute (CCSB-DFCI). horfeome v8.1 has undergone more extensive QC than previous versions, having had clonal isolates verified by next-gen sequencing. Altogether, this collection represents almost 12, unique genes. Originally derived from fullysequenced MGC cdna clones, these fully sequenced ORFs have been Gateway-adapted to enable efficient transfer of the ORF construct into compatible expression vectors. Fully-sequenced human ORF collection Gateway-entry format for efficient recombination Stop codons have been removed to allow for fusion proteins Product details attr1 puc Origin ORF pdonr223 SpectR attr2 Pre-made cdnas and ORFs II.2 The ORFeome Collaboration Collection The ORFeome Collaboration (OC) is a group of academic and commercial laboratories that was formed to develop an unrestricted source of sequence-validated human ORF clones. Using the MGC cdna clones as starting material, the OC has been able to create over 1, full-length human ORF clones, making significant progress toward the goal of providing at least one ORF for each of the ~18,5 defined human genes. GE Healthcare is a major distributor for the OC, providing individual clones, gene family libraries and whole-genome collections to academic and commercial customers. The use of the Gateway recombinational cloning system in all of the OC ORF clones allows for efficient transfer of the ORFs from one vector to another, providing the researcher with a broad choice of destination vectors. Furthermore, OC ORFs are available with or without native stop codons, providing options for native and fusion-ready protein expression. Fully-sequenced, genome-scale ORF collection Easily transferable into any Gateway-adapted expression vector Available with or without native stop codons for native and fusion-ready expression Product details att ORF att Individual clone glycerol stock Libraries: pre-defined gene families or custom rearray glycerol stock Genome-scale library glycerol stock Catalog No. ORFeome Collaboration Collection Human ORFeome Collaboration Clone (with native stop codon) - glycerol Human ORFeome Collaboration Clone (without stop codon) - glycerol ORFeome Collaboration Mouse Clone (without native stop codon) glycerol ORFeome Collaboration Mouse Clone (with native stop codon) glycerol OHS5893 OHS5894 OMM5896 OMM5895 II.2 Pre-made cdnas and ORFs Vector Element Utility ORF Human ORFs from fully sequenced MGC clones attr1/attr2 Geteway recombination sites for easy subcloning and transfer ORF clones are derived from cdna clones by removing the untranslated region (UTR), leaving just the protein coding sequence known as ORF. Related products Precision LentiORF Clones and Libraries See p. 87 Easily transfer your gene of interest to an expression vector for any application Gene Family Libraries See p. 88 Transmembrane Receptor Transporter Translation Regulator Transcription Regulator Phosphatase Protease Rest of Genome Ligand-Dependent Nuclear Receptor att att Entry Clone ORF Destination Clone att att TurboFECT Transfection Reagent See p. 89 Clones are available with and without stop codons Kinase Ion Channel Growth Factor Human ORFeome V8.1 - Function by Gene ID Schematic showing the proportions of gene coverage based on the known function of each Gene ID G-Protein Coupled Receptor Enzyme Cytokine att Phenotype ORF Expression Clone Localization att Purification Schematic showing the transfer of ORF content to Gateway-adapted expression vector Gateway cloning provides fast and efficient gene content portability without the need for PCR or re-sequencing. This allows flexible cloning strategies to quickly incorporate the cloned ORF into many different experimental designs. Pre-made cdnas and ORFs Pre-made cdnas and ORFs 84 85

45 Pre-made cdnas and ORFs II.2 CCSB-Broad Lentiviral Expression Library Individual clone glycerol stock Libraries: pre-defined gene families or custom rearray glycerol stock Genome scale library glycerol stock Related products Catalog No.* CCSB Human ORFeome Collection CCSB-Broad Lentiviral OHS685 Expression Clone CCSB-Broad Lentiviral OHS693 Expression Library CCSB-Broad Lentiviral Expression Clone Mutant OHS6269 CCSB-Broad Lentiviral Expression Clone OHS6271 Partially Sequenced CCSB-Broad Lentiviral Expression Clone OHS627 Unsequenced TurboFECT Transfection Reagent See p. 89 Gene Family Libraries See p. 88 Schematic of the creation of the CCSB- Broad lentiviral expression library The available collection of MGC cdnas cdnas transferred to Gateway ORF templates in polyclonal by PCR. Clonal plasmid isolates were derived from single bacterial colonies, sequenced, and filtered to determine clones to be included in horfeome V8.1 collection. Using Gateway recombination, the entry clone collection was transferred into the PLX34-Blast-V5 destination vector. A single colony was isolated from each recombination reaction, resulting in the CCSB-Broad Lentiviral Expression Library. The CCSB Broad Lentiviral Expression Library is a genome-scale expression collection developed by researchers at Dana-Farber Cancer Institute and The Broad Institute to provide a sequence confirmed collection of human ORFs in an expression-ready lentiviral system. This library was derived from the horfeome V8.1 and created to enable targeted experiments and large-scale screening in diverse cell types. CCSB-Broad Lentiviral Expression Clones (OHS685) are fully sequenced, and match their accessions with no more than a single mutation within the open reading frame Lentiviral expression vector allows creation of lentiviral particles for transduction of almost any mammalian cell type CMV promoter drives strong expression of the ORF construct Blasticidin resistance allows for multiplex experiments in cells containing Puromycin or Neomycin resistance V5 tag facilitates pull-downs and co-immunoprecipitation expereiments in order to determine protein-protein interactions, also allows for labeling of expressed ORFs in cells via immunofluorescence Product details PCR to add Gateway sequences to ORF templates from the MGC ORF 5' LTR Ψ A B C D E F G RRE PKG puc ori Bsd R horfeome V Arrayed Gateway transfers (LR) into plx34-blast-v5 expression vector plx34-blast-v5 Vector Element Utility PGK promoter Human phosphoglycerate kinase (PGK) promoter drives expression of BsdR gene BsdR Blasticidin resistance for antibiotic selection of transduced cells hcmv Promoter Human cytomegalovirus promoter drive transgene expression ORF Open Reading Frame coding protein of interest V5 V5 Epitope Tag for protein analysis of transgene product 3 SIN-LTR 3' Self inactivating long terminal repeat for increased lentiviral safety 5 LTR 5' long terminal WPRE Woodchuck hepatitis posttranscriptional regulatory element enhances transgene expression in the target cells cppt/cts Central polypurine tract increases translocation into the nucleus of non-dividing cells RRE Rev response element enhances titer by increasing packaging effeciency of full-length viral genomes Amp R hcmv ORF V5 stop F1 ori Pick one colony from each LR reaction WPRE 3' SIN LTR A B C D E F G CCSB-Broad Lentiviral Expression Library II.2 Pre-made cdnas and ORFs Precision LentiORF Collection The Dharmacon Precision LentiORF Collection is a library of full-length ORFs in an expressionready lentiviral vector generated from the fully sequenced ORFeome Collaboration Collection. These ready-to-use ORFs are high-quality, versatile and guaranteed* to express. Available as plasmid or high-titer viral particles for difficult-to-transfect cells Efficient expression even at low multiplicity of infection (MOI)* Multi Tag cloning site allows for addition of custom fusion tags. Functional data For easy-to-transfect cells Create your own lentiviral particles for difficult-totransfect cells 5' LTR Ψ Amp R MOI MOI MOI MOI hcmv ORF IRES Multi Tag Cloning Site puc ori ploc Precison LentiORF Starter Kits 2 ORF clones Glycerol Transfection Starter Kit RFP control Glycerol TurboFect Transfection Reagent 2 ORF clones Glycerol Transduction Starter RFP control Glycerol Kit Calcium Phospate Transfection Reagent Trans-Lentiviral packaging mix (1 rxn) Transduction-ready 2 ORF clones high-titer virus, TU/mL high-titer lentiviral particles Viral particle Starter Kit RFP control high-titer virus, TU/mL -2atGFPnuc Viral particles per cell ORF Tubulin SV4 ori Vector Element Utility hcmv Human cytomegalovirus promoter drives strong transgene expression ORF Full-length human ORFs from the ORFeome Collaboration Collection Multi Tag Cloning Site Convenient cloning site for the addition of a purification or tracking tag IRES Internal ribosomal entry site allows expression of TurboGFP and puromycin resistance genes in a single transcript tgfpnuc TurboGFP reporter expressed in the cell nucleus to facilitate visual tracking of transduction and expression Blast R Blasticidin resistance permits antibiotic-selective pressure and propagation of stable integrants 2a 2a self-cleaving peptide allows simultaneous expression of tgfpnuc and blasticidin resistance proteins from a single transcript 5' LTR 5' long terminal repeat 3' SIN LTR 3' self-inactivating long terminal repeat for increased lentivirus safety Ψ Psi packaging sequence allows viral genome packaging using lentiviral packaging systems WPRE Woodchuck hepatitis posttranscriptional regulatory element enhances transgene expression in the target cells Blast R WPRE 3' SIN LTR Controlled ORF expression levels with increasing viral particles HEK293T cells transduced with a lentiviral ORF at a range of MOIs. Western blot analysis depicts an increase in protein expression correlating with an increase in the number of LentiORF viral particles applied to the cell. Individual clone glycerol stock or viral particles Libraries: pre-defined gene families or custom rearray glycerol stock Genome-scale library glycerol stock Precision LentiORF RFP Controls Precision LentiORF individual clone (with native stop codon) - glycerol Precision LentiORF individual clone (without stop codon) - glycerol Precision LentiORF individual clone (with native stop codon) - viral particles Precision LentiORF individual clone (without stop codon) - viral particles Catalog No.* OHS5897 OHS5898 OHS5899 OHS59 Precision LentiORF RFP Controls Glycerol stock Viral particles Precision LentiORF Starter Kits Transfection Starter Kit Transduction Starter Kit Viral particle Starter Kit OHS5832 OHS5833 OHS5834 OHS5835 OHS5836 * When used as part of a LentiORF starter kit according to kit protocols. Related products GFP TurboFECT Transfection Reagent See p. 89 Trans-Lentiviral ORF Packaging Kit See p. 91 II.2 Pre-made cdnas and ORFs Pre-made cdnas and ORFs Pre-made cdnas and ORFs 86 87

46 Pre-made cdnas and ORFs II.2 Pre-defined Gene Family cdna and ORF Libraries Libraries: pre-defined gene families or custom rearray glycerol stock Related products CCSB Human ORFeome Collection See p. 83 Mammalian Gene Collection See p. 83 CCSB-Broad Lentiviral Expression Library see p. 86 TurboFECT Transfection Reagent See p. 89 Pre-defined cdna and ORF libraries have been assembled using bioinformatic tools to reflect genetic and ontological information from peer-reviewed publications and databases. MGC cdnas are full-length and fully-sequenced, 1 and are recognized as the gold standard for cdna clones Expression-Ready cdnas are a subset of the MGC that have been cloned into a mammalian expression vector Human ORFeome clones contain Gateway att sites to enable the transfer of open reading frames to expression vectors; native stop codons have been removed CCSB-Broad Lentiviral Expression Library clones are expression-ready human ORFs derived from the horfeome v8.1 collection that have been cloned into a lentiviral expression vector TurboFect Transfection Reagent TurboFect Transfection Reagent is a highly efficient, easy-to-use, transfection reagent based on a novel, proprietary, cationic polymer. TurboFect Transfection Reagent has been shown to be non-immunogenic and non-toxic in all cells tested. Ideal for transfection of a variety of cells, including primary, differentiated and undifferentiated cells Transfection can be performed in the presence or absence of serum Demonstrates superior transfection efficiency and minimal toxicity when compared to lipidbased or other polymer-based transfection reagents Simple 3-step transfection protocol compared with 6-7 steps for other transfection reagents Functional data TurboFect Cell Line Cell Type % MFI Viability % Catalog No. TurboFect Transfection Reagent 1 ml (sufficient for 3-5 transfections) in 96-well format R531 5 x 1 ml R532 Related products Precision LentiORF Clones and Libraries See p. 87 CCSB-Broad Lentiviral Expression Library see p. 86 II.2 Pre-made cdnas and ORFs COS-7 African green monkey kidney cells 98 2,29 9 Contact Technical Support to build your custom gene set Pre-defined Gene Expression Libraries Fully-sequenced cdna Sets Expression-Ready cdna Sets HUMAN (number of genes) CCSB Human ORFeome Sets CCSB-Broad Lentiviral Library Sets Cell Cycle Cytokine GPCR HeLa Human cervix adenocarcinoma cells 97 2,21 95 CHO Chinese hamster ovary cells 89 1, HEK293 Human embryonic kidney cells 94 1,224 8 B5 Rat nervous tissue neuronal cells 6 1, Calu1 Human lung epidermoid carcinoma cells RAW264 Mouse leukaemic monocyte-macrophage cells A Growth Factor WEHI Mouse B cell lymphoma cells Insulin Receptor Signaling 27 MDCK Madin Darby Canine Kidney cells 92 1,1 94 Ion Channel COLO Human colon adenocarcinoma cells Kinases Nuclear Receptor Sp2/Ag14 Mouse myeloma cells HeLa S3 Human cervix carcinoma cells B Oxygen Binding 26 Hep2C Human larynx carcinoma cells Peptidase L929 Mouse connective tissue fibroblasts Phosphatases NIH/3T3 Mouse embryo fibroblasts 97 2,4 89 Plasma Membrane 2, ,24 HLF Primary human lung fibroblasts Protein Kinase Transcription Factor Transporter 1, Ubiquitin High transfection efficiency of a wide variety of cell types. Cell lines were transfected with plasmid DNA coding for egfp. The transfection was evaluated by flow cytometry for percentage (%) of cells expressing egfp Mean Fluorescence Intensity (MFI), which reflects the levels of egfp expression in the transfected cells and cellular viability. Successful transfection of primary cells. Transfection of mouse bone marrowderived dendritic cells (A) and macrophages (B) with TurboFect reagent. Gene number may fluctuate as libraries undergo revision or when gene records are updated. References 1. MGC (Mammalian Gene Collection) Program Team (22) Generation and initial analysis of more than 15, full-length Human and Mouse cdna sequences. PNAS 99(26): Triple co-transfection of HeLa cells with TurboFect Transfection Reagent. HeLa cells were co-transfected with 1:1:1 mixture of plasmids encoding for enhanced green fluorescent protein localized in the cytoplasm, enhanced cyan fluorescent protein (egfp) with nuclear localization signal and monomeric DsRed protein with membrane localization signal. Pre-made cdnas and ORFs Pre-made cdnas and ORFs 88 89

47 Pre-made cdnas and ORFs II.2 TurboFect in vivo Transfection Reagent Catalog No. TurboFect in vivo Transfection Reagent Size: 12 µl Concentration: 2 transfections using 5 µg DNA Related products R541 Precision LentiORF Clones and Libraries See p. 87 Studies that include nucleic acid delivery to live animals require reagents that will minimize cellular toxicity and inflammation while maximizing delivery efficiency. TurboFect in vivo Transfection Reagent has been found to be highly effective for DNA plasmid delivery in vivo, while not inducing an inflammatory response. No detectable inflammatory response Gene delivery via multiple routes of administration Functionally tested in mice and rats Functional data 1,4 1,2 1, Negative control (DNA only) Positive control (LPS) TurboFect in vivo Tranfection Reagent Trans-Lentiviral ORF Packaging Kit Trans-Lentiviral ORF Packaging Kit allows the researcher to generate lentiviral particles containing gene expression constructs. The resulting viral particles can be used to transduce cells of interest for long-term ORF expression. Effective packaging of second- or third-generation lentiviral vectors Genes encoding the components required for packaging the viral genome are separated across five plasmids, minimizing the chance of recombinant replication-competent virus production Generate lentiviral particles that effectively transduce both dividing and non-dividing mammalian cells in vitro and in vivo Titers of TU/mL can be achieved and virus can be further concentrated up to TU/mL Available in convenient 1, 5 and 1 reaction sizes Packaging Mix Transfer Vector Catalog No. Trans-Lentiviral ORF Packaging Kit with calcium phosphate 1 reactions TLP reactions TLP reactions TLP592 Trans-Lentiviral ORF Packaging Kit with calcium phosphate and HEK293T 1 reactions TLP5918 Calcium Phosphate Transfection Reagent Kit 5 reactions TLP5915 HEK293T cell line 1 ml HCL4517 II.2 Pre-made cdnas and ORFs TNFα (pg/ml) Related products Precision LentiORF Clones and Libraries See p. 87 hours 1 hour 3 hours 6 hours 24 hours No prolonged inflammatory response after DNA delivery using TurboFect in vivo Transfection Reagent. 5 µg of plasmid DNA was delivered into female BALB/c mice via intravenous injection using TurboFect in vivo Transfection Reagent. The concentration of proinflammatory cytokine TNFα was determined by ELISA in blood serum samples taken at the indicated time points. (LPS; lipopolysaccharides). Transfect cells with packaging plasmid and transfer vector containing ORF Isolate replication-incompetent viral supernatant LUC RLU/mg of protein 12, 1, 8, 6, 4, 2, LUC RLU/mg of protein 5, 4, 3, 2, 1, TurboFect in vivo Tranfection Reagent ExGen 5 in vivo Tranfection Reagent Vendor A Vendor B Negative control (DNA only) lungs heart liver spleen kidney ovary Comparison of TurboFect in vivo Transfection Reagent with other in vivo transfection reagents. 5 µg of plasmid DNA coding for firefly luciferase was complexed with various in vivo transfection reagents and delivered into female BALB/c mice via intravenous injection. Transfections were performed according to manufacturer's recommendations. Firefly luciferase expression was determined in the indicated organs 24 hours after plasmid DNA delivery. Schematic showing production of lentiviral particles using the Trans-Lentiviral Packaging System. Co-transfection of the packaging plasmids and transfer vector into the packaging cell line, HEK293T, allows efficient production of lentiviral supernatant. Virus can then be transduced into a wide range of cell types, including both dividing and non-dividing mammalian cells. Kit Contents Trans-Lentiviral ORF Packaging Kit with Calcium Phosphate TLP5916 (1 reactions) TLP5918 (1 reactions + cells) TLP5919 (5 reactions) TLP592 (1 reactions) Trans-Lentiviral Packaging Mix 285 μg 285 μg 285 μg μg 1 HEK293T Packaging Cell Line not included 1. ml not included not included Calcium chloride reagent 1.2 ml 1.2 ml 6 ml 6 ml 2 2X HBSS reagent 12 ml 12 ml 6 ml 6 ml 2 Precision LentiORF RFP control DNA 45 μg (.45 μg/μl) 45 μg (.45 μg/μl) not included not included Pre-made cdnas and ORFs Pre-made cdnas and ORFs 9 91

48 Non-mammalian Resources II.3 Non-mammalian Resources for Comprehensive Analysis Processes relevant to human biology can be studied in many other organisms. The conservation of biological processes and the common descent of all organisms allows scientists to overcome many practical and ethical barriers by taking advantage of the benefits of model organisms. Thus, a range of model organisms have contributed significantly to scientific discovery. In fact, the inherited traits of peas gave Gregor Mendel 1 the first true insight in human genetics. Today the most cited model organisms used to study human biology are Mus musculus, Saccharomyces cerevisiae, Escherichia coli, Drosophila melanogaster, Caenorhabditis elegans and Danio rerio (zebrafish). Each has facilitated significant breakthroughs and each continues to play an important role in an era of omics. Perform comprehensive research of basic gene function in model organisms page 94 Multiple yeast collections for expanding your research High-quality cdna and ORF clones and collections from multiple non-mammalian species page 94 Perform whole genome RNAi screens in C. elegans II.3 Non-mammalian Resources choosing the right organism turned out to be as important as having addressed the right problems to work on. - Sydney Brenner, Nobel Lecture 22 pages Section II Chapter 3: Table of Contents page 96 Model organism selection guide Yeast Collections Molecular Barcoded Yeast ORFs E. coli Collections C. elegans Collections Xenopus Gene Collection Danio rerio (Zebrafish) Gene Collection References 1. Mendel, G. (1866) Experiments on Plant Hybridization [Versuche uber Pflanzen-Hybriden]. Verh. Naturforsch. Ver. Brunn 4:3-47 (in English in 191, J. R. Hortic. Soc. 26:1-32). Non-mammalian Resources Non-mammalian Resources 92 93

49 Non-mammalian Resources II.3 Which model organism and product format apply to your research? Saccharomyces cerevisiae (Yeast) Caenorhabditis elegans Escherichia coli RNAi Knockouts ORFs Tagged ORFs cdnas Promoters Genomic DNA Format Yeast Collections Yeast Knockout Strains The Saccharomyces Genome Deletion Project 1 created over 6, gene-disruption mutants* that cover 96% of the yeast genome providing a unique tool for functional analysis. Molecular barcodes that identify each strain allows phenotypic analysis to be performed on a single gene basis or on a genome-wide scale. 2 Yeast Knock Out MATa Collection Yeast Knock Out MATalpha Collection Yeast Knock Out Heterozygous Collection Yeast Knock Out Homozygous Diploid Collection Yeast Knock Out Essential Genes Collection Catalog No. YSC153 YSC154 YSC155 YSC156 YSC157 Individual clone glycerol stock Genome-scale library glycerol stock Custom rearray glycerol stock * While > 6, represents the number of individual genes, there are four backgrounds (haploids of MATa and MAT alpha, heterozygous diploid, and homozygous diploid) with many genes available in more than one background, thereby creating over 2, knock out strains. II.3 Non-mammalian Resources Danio rerio (Zebrafish) Xenopus sp. Yeast TAP-Tagged Collection For accurate profiling of the yeast proteome, we offer the Yeast TAP-Tagged Collection. This collection includes more than 4, fusion-tagged ORFs under control of the endogenous promoter at their original chromosomal location. 3 Through immunodetection of the common tag, researchers can create a census of proteins expressed during log-phase growth and quantify their absolute levels. Individual clone glycerol stock Genome-scale library glycerol stock Follow the icons to your product solution Glycerol stock: Bacterial culture transformed with a plasmid vector, supplied in glycerol-containing medium for freezer storage and supplied in single tubes. The Yeast-TAP-Fusion Library allows the purification and selection of the yeast proteome and associated components by using two affinity selection steps in tandem, enabling the development of a range of high throughput functional assays. TAP-Tagged ORF Collection TAP-Tagged strain (endogenous promoter) Yeast TAP-Fusion Membrane Protein Subset Catalog No. YSC1177 YSC1178 YSC1247 Arrayed format: Genome-scale collections or custom rearrays of bacterial or yeast glycerol stocks arrayed in 96-well microtiter plates. Yeast Tet-Promoter Hughes Collection (ythc) The ythc collection contains 8 essential yeast genes whose endogenous promoter has been replaced with an inducible promoter. 4,5,6 The expression of the gene can be switched off by the addition of doxycycline to the yeast s growth medium, thereby overcoming the limitation of working with an essential gene. Yeast Tet-promoters Hughes Collection (ythc) Collection Catalog No. YSC1182 Individual clone glycerol stock Genome-scale library glycerol stock Custom rearray glycerol stock Yeast Tet-promoters Hughes (ythc) Strain YSC118 Yeast Tet-promoters Hughes Parental Strain R1158 YSC121 References 1. Winzeler, E.A. et al. (1999) Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science 285: Giaever, G. et al. (22) Functional profiling of the Saccharomyces cerevisiae genome. Nature 418: Ghaemmaghami, S. et al. (23) Global Analysis of Protein Expression in Yeast. Nature 425: Winzeler, E.A. et al. (1999) Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel analysis. Science 285: Mnaimneh, S. et al. (24) Exploration of essential gene functions via titratable promoter alleles. Cell 118(1): Hughes, T.R. et al. (2) Functional discovery via a compendium of expression profiles. Cell 12(1): Non-mammalian Resources Non-mammalian Resources 94 95

50 Non-mammalian Resources II.3 Molecular Barcoded Yeast (MoBY) ORFs Pooled collection glycerol stock Entire collection glycerol stock Custom rearray glycerol stock Individual clones Catalog No. Molecular Barcoded Yeast (MoBY) ORFs Molecular Barcoded Yeast (MoBY) ORF Collection YSC5432 The MoBY ORF Collection enables simultaneous measurement of all transformants in a single pooled culture using a barcode microarray readout. This genome-scale tool can be used in a broad range of experiments from mutation screening to mode-of-action studies for drug discovery. 1 Features Genome-scale collection of > 4, uniquely barcoded ORFs Dual barcodes provide increased hit confidence Native context facilitates sequencing upstream and downstream of the ORF An endogenous promoter provides biologically relevant expression levels E. coli Collections E. coli Keio Knockout Strains For systematic loss-of-function analysis of the E. coli genome, the E. coli Keio Knockout Collection contains deletion strains for all non-essential genes in E. coli. 1,2 There are two independent deletion strains for each of 3,985 genes, yielding 7,97 strains. Each deleted gene is replaced with a kanamycin resistance cassette that can be excised by FLP recombination. This collection may be used for systematic analyses of unknown gene function and for genome-wide testing of mutational effects. Number of genes 3,985 Number of strains 7,97 Background E. coli K-12 BW25113 Individual strain glycerol stock Collection glycerol stock Custom rearray glycerol stock References 1. Baba, T. et al. (26) Construction of Escherichia coli K12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2: Datsenko, K.A. and Wanner, B.L. (2) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS 97: II.3 Non-mammalian Resources Catalog No. Collection OEC4988 Schematic of a pooled screen using the MoBY ORFs Each ORF in the MoBY collection is paired with a unique dual barcode cassette allowing pooled screening and are expressed using their endogenous promoter to mimic native expression levels. The figure outlines a schematic of a pooled drug selection screen. (Upper) A pool of barcoded ORF clones are transformed into a drug-resistant yeast strain and split into experimental groups. (Lower) Expression of one of the ORFs causes a reversion back to the wildtype, drug-sensitive phenotype. Comparison of the experimental groups shows a significant decrease in the barcode associated with this gene in the treated pool versus the untreated pool. Untreated Endogenous Promoter ORF Drug-resistant unknown mutation Treated UTR Dual Barcodes BC1 BC2 Barcoded ORFs are pooled Pooled ORFs are transformed into drug-resistant mutant Transformants split into experimental groups Expressing the ORF responsible for resistance to the drug re-sensitizes cells to the treatment Fitness changes can be measured simultaneously within the heterogenous population using a microarray to detect barcodes E. coli Keio Knockout Strain OEC4987 BW25113 Parental Strain OEC542 E. coli Promoter Collection This collection of E. coli strains enables monitoring of gene expression at high temporal resolution in living cells. 3,4 Each of the reporter strains has a bright, fast-folding green fluorescent protein (GFP) fused to a full-length copy of an E. coli promoter in a low copy plasmid, enabling measurement of gene expression at a resolution of minutes with high accuracy and reproducibility. Number of promoters 1,9 Strain Promoter Strain Promoter Collection E. coli K-12 MG1655 Catalog No. PEC3876 PEC3877 Individual clone glycerol stock Collection glycerol stock Custom rearray glycerol stock References 3. Kalir, S. et al. (21) Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292: Ronen, M. et al. (22) Assigning numbers to the arrows: parameterizing a gene regulation network by using accurate expression kinetics. PNAS 99: Additional Yeast Collections Product Catalog No. Yeast ORF Collections YSC3867, YSC3868, YSC3869, YSC387, YSC388 Tagged ORF Clones and Strains HA-tagged Collections YSC168, YSC17, YSC1764 GST-tagged Collections YSC4515, YSC4423 Yeast Decreased Abundance (DAmP) YFP-tagged Collections Haploid DAmP Strains & Collections Diploid DAmP Strains & Collections YSC518 YSC59, YSC594 YSC55, YSC593 Genomic DNA Genomic Tiling Strains & Collections YSC4613, YSC513, YSC534 Protein-protein Interaction Strains Yeast Interactome Collection YSC5849, YSC597, YSC598 E. coli Tagged ORFs This collection of SPA- and TAP-tagged E. coli strains facilitate mapping of protein-protein interactions, offering insight into the function of uncharacterized proteins and the study of topological organization for the bacterial interactome. 5,6 Number of tagged proteins 1,9 Catalog No. E. coli SPA-tagged Strain OCE4626 E. coli SPA-tagged Collection OEC4627 E. coli TAP-tagged Strain OEC4629 E. coli TAP-tagged Collection OEC463 Individual clone glycerol stock Collection glycerol stock Custom rearray glycerol stock References 5. Butland, G. et al. (25) Interaction network containing conserved and essential protein complexes in Escherichia coli. Nature 433: Zeghouf, M. et al. (24) Sequential peptide affinity (SPA) system for the identification of mammalian and bacterial protein complexes. Journal of Proteome Research 3: Ho, et al. (29), A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds. Nature Biotechnology 4: Non-mammalian Resources Non-mammalian Resources 96 97

51 Non-mammalian Resources II.3 C. elegans Collections Individual clone glycerol stock Collection glycerol stock Custom rearray glycerol stock C. elegans ORFs The C. elegans ORF collection is the leading resource for functionally characterizing the worm proteome through applications such as protein-protein interactions, RNAi and protein expression. C. elegans ORF clones can be subcloned into any expression vector, using traditional restriction enzyme and ligase cloning methods. Number of genes > 1, Xenopus Gene Collection (XGC) The Xenopus Gene Collection (XGC) provides clones that are fully sequenced and verified to contain a complete coding sequence (CDS) of expressed genes from Xenopus laevis and Xenopus tropicalis. Xenopus sp. Collection X. laevis X. tropicalis XGC Full-ORF Clones 1,321 8,318 Individual clone glycerol stock Custom rearray glycerol stock Reference II.3 Non-mammalian Resources Catalog No. Non-redundant Genes 9,524 7,362 Collection OCE4518 C. elegans ORF Clone OCE1182 XGC Fully Sequenced Clone Catalog No. MXL1736 Individual clone glycerol stock Collection glycerol stock Custom rearray glycerol stock C. elegans ORF-RNAi Collection The C. elegans ORF-RNAi collection includes clones containing full-length open reading frames for over 11, genes ready for RNAi screening. The ORF containing the full coding region is cloned into an RNAi feeding vector and compatible host. Plasmid preparations of the ORF-RNAi clones can be used as templates for in vitro double-stranded RNA synthesis for soaking or injection. Number of genes > 11, Bacterial feeding strain Collection HT115(DE3) Catalog No. RCE1181 XGC Fully Sequenced Xenopus tropicalis cdna clone IMAGE Xenopus cdna Clone Zebrafish Gene Collection MXT1765 EXL151 Zebrafish cdna and ORF collections include over 16, fully-sequenced ORFs from the Zebrafish Gene Collection (ZGC). Exclusive access to Exelixis Zebrafish cdna Collection and unique Zebrafish EST cluster from Singapore s Institute for Molecular and Cell Biology (IMCB) is also provided. Individual clone glycerol stock Custom rearray glycerol stock C. elegans RNAi V1.1 Clone RCE1182 Zebrafish Collection Content Individual clone glycerol stock Collection glycerol stock Custom rearray glycerol stock C. elegans Promoters The C. elegans Promoter Collection includes clones containing the predicted promoters for over 6, genes designed to easily construct GFP reporter strains, identifying when and where genes are expressed. Number of genes > 6, ZGC full ORF Clones IMAGE Zebrafish cdna Clones Exelixis Zebrafish cdna Clones IMCB Zebrafish ESTs > 16, Full ORF Clones 432, Searchable ESTs 18, Searchable ESTs 15,59 Unique EST Clusters Catalog No. Catalog No. ZGC Fully Sequenced Zebrafish cdna MDR1734 Collection PCE1181 Zebrafish cdna clone EDR152 Promoter Clone PCE1182 Zebrafish EST cdna (Exelixis) EDR4422 IMCB Zebrafish EST cdna MDR1738 C. elegans Transcription Factors Yeast and E. coli Individual clone or strain glycerol stock Collection glycerol stock Custom rearray glycerol stock One (Y1H) and two (Y2H) hybrid assays are gene-centered alternatives to the more traditional chip on HhIP approach to the mapping of transcription factor regulatory networks. The C. elegans Transcription Factor Collection consists of 755 C. elegans transcription factor ORFs fused with the GAL4p activation domain and cloned into the yeast prey vector pdest AD. This collection is available in either Yeast or E. coli. Number of genes (ORFs) 755 Catalog No. C. elegans Transcription Factors Collection (E. coli ) OCE4819 C. elegans Transcription Factors Collection (Yeast) OCE4821 C. elegans Transcription Factor Clone (E. coli) OCE4818 C. elegans Transcription Factor Strain (Yeast) OCE482 Non-mammalian Resources Non-mammalian Resources 98 99

52 Frequently asked questions (FAQs) Dharmacon products Appendix Frequently asked questions (FAQs) shrna microrna Custom oligonucleotide synthesis Gene expression Trademarks and license information Glossary Index by term Index by product name QUESTIONS What is the difference between sigenome and ON-TARGETplus? What is the difference between a SMARTpool, the Set of 4 and the Set of 4 Upgrade? Do you have pre-designed s against my gene of interest? What are the recommended controls for my experiment? ANSWERS The genome-wide collection of ON-TARGETplus includes a patented dual-strand modification pattern to decrease off-targets by up to 9%. Unlike common modifications that only block the sense strand, ON-TARGETplus also includes a modification to the antisense seed region, which is shown to be a primary cause of microrna-like off-target activity. sigenome remains an excellent choice for high-efficiency gene silencing, but does not carry the specificity-enhancing modifications. Both sigenome and ON-TARGETplus products carry the same functional guarantee of 75% silencing or better by the SMARTpool and at least three of the four individual s at the mrna level with appropriate controls. We do encourage new research projects and new customers to begin with the ON-TARGETplus platform to have the advantage of the performance-enhancing modifications. The sigenome products are available for ongoing experiments and for studies which require the use of unmodified s. Within each product line, we pre-design four different s for each human, mouse or rat target. The SMARTpool products contain all four s pooled together in a single tube. Pooling rationally designed s leads to decreased off-target effects. In addition, a SMARTpool will typically silence as well as the best in the pool, saving you time in screening through multiple s to achieve a high degree of silencing. The Set of 4 contains all four s provided in separate tubes. In this format, silencing of each can be assessed separately. The Set of 4 Upgrade is the same product at a decreased price which requires a prior or concurrent purchase of the SMARTpool for the same target. We also offer each for individual purchase. Sequence information is provided with the purchase of all of our pre-designed products. We have pre-designed s for nearly all human, mouse and rat protein-coding genes in the RefSeq database. To find pre-designed s for your gene of interest, first initiate a search for your gene on gelifesciences.com/dharmacon. You can use the gene name, gene symbol, gene ID or RefSeq accession number as a search term. Once your search results are displayed, click on the desired gene to view a list of products available for that target. You are encouraged to review the detailed gene information at the top of the page and use the gene ID link to view the NCBI Entrez Gene page, to confirm that you have identified the correct target gene. A non-targeting control is designed to have no known target in the cells being used. These negative controls are important for distinguishing sequence-specific silencing from non-specific effects in the RNAi experiment. Because non-specific effects can be concentration dependent, it is important to use the negative control at the same concentration as your gene-specific. Efficient delivery of is critical for achieving significant gene silencing. A positive control can be used to optimize delivery conditions and to monitor transfection efficiency throughout your experiments. An extensively validated that targets a housekeeping gene is ideal as a positive control. We offer several positive and negative controls in our Accell, ON-TARGETplus and sigenome product lines as well as fluorescent controls for your experiments. To learn more please refer to our Technical Note: Effective Controls for RNAi Experiments or see page 56 to review our complete offering. Continued on next page. 1 Appendix Appendix 11

53 Dharmacon products continued. Dharmacon shrna products QUESTIONS ANSWERS QUESTIONS ANSWERS What is Accell? How is the Accell delivery protocol different from standard delivery protocols? Accell is specially modified to enable delivery into difficult-to-transfect cell types without the use of a transfection reagent, virus or other chemical or physical methods of delivery (such as electroporation). In addition to enabling uptake, the modifications stabilize the against endo- and exonuclease degradation, making them ideal for in vivo RNAi experiments. There are over 1 citations demonstrating effective silencing from Accell in neuronal, immunological, primary, differentiated and other difficult-totransfect cell types. The primary differences that should be noted for the Accell delivery protocol are the following: Accell should be delivered at a 1 μm concentration (optimization recommended) Cell culture conditions during Accell application should be no-serum or low-serum (< 2.5%; optimization is highly recommended) mrna knockdown is typically measured at 72 hours, protein at 96 hours What is the difference between a simple stem-loop shrna and a microrna-adapted shrna? Which shrna products are validated or provided with a guarantee? A simple stem-loop shrna construct is a standard hairpin (basic stem-loop-stem structure) that is delivered via transfection or transduction to your cells. Each of the constructs in the GIPZ, TRIPZ, SMARTvector and shmimic microrna collections incorporate stem-loops that are flanked by microrna sequences. For this reason, they are referred to as microrna-adapted constructs. For GIPZ and TRIPZ, microrna-3 (mir-3) sequences are used; for SMARTvector and shmimic micrornas, a proprietary microrna scaffold is used. Adding this microrna-based scaffold sequence to the shrna construct is thought to provide Drosha and Dicer cleavage sites, thereby facilitating a more efficient hand off through the endogenous RNAi pathway. Dharmacon shrna constructs are not individually validated, as we find that shrna efficiency varies depending on which cell line you are using. Additional factors such as appropriately optimized transfection/transduction conditions may also contribute to shrna efficacy. Generally, for microrna-adapted shrnas we observe one out of three shrnas provide at least 7% or greater knockdown at the mrna level with appropriate controls. For TRC (plko.1 vector), we have observed that one to two out of five TRC shrna clones per gene will give at least 7% knockdown. Can I substitute any delivery media for the Accell Delivery Media? Accell Delivery Media provides the conditions proven to be optimal for Accell application. However, the use of other serum-free media, or Accell Delivery Media with supplemental serum or growth factors, continue to demonstrate successful results. For additional information, please contact our Technical Support team. When would it be necessary to request a custom instead of a pre-designed? Can I get expert help designing my custom? Which transfection reagent should I use with my specific cell line? There are four pre-designed s for every gene in human, mouse and rat sold as catalog items for the sigenome, ON-TARGETplus and Accell product lines. If additional requirements exist for targeting alternative species, individual splice variants, specific gene regions or multiple family members, custom synthesis of your will be necessary. Some researchers also request a custom sequence to replicate a published result. Additionally, there are numerous options available to customize an (catalog or custom) with chemical modifications or special processing. Custom can be ordered online up to the 1 µmol scale at gelifesciences.com/dharmacon/rnai-and-custom-rna-synthesis. For additional information, please contact our Technical Support team. If you would like assistance in designing your custom, please contact our Technical Support team. Alternatively, we also provide a free, online design tool at our sidesign Center gelifesciences.com/dharmacon/ design-center. The sidesign tool uses the proprietary SMARTselection algorithm for rational design. The best transfection reagent will depend on the cell line that you are using and must be determined empirically, as no single reagent is optimal for all cell types. We offer the DharmaFECT Transfection Reagents - four distinct lipid-based transfection reagents that have been specially formulated for delivery of s into cells. DharmaFECT 1 is the most all-purpose transfection reagent, but in many cell types, DharmaFECT 2, 3 or 4 improves overall efficency and viability. Please consult the online Support Center for optimized conditions for over 4 common cell lines or see page 61 for the Cell Type Guide. If your cell line is not identified in the compatibility table or DharmaFECT Cell Type Guide and you are not able to find an example of delivery in this cell line in published literature, we recommend testing the Set of 4 DharmaFECT reagents to identify the best one for your experiments. The optimal transfection reagent is one that results in a high level of delivery with a low level of cell toxicity. The recommended method for confirming delivery is to assay knockdown, preferably at the mrna level, by a positive control that targets a housekeeping gene for which silencing is well characterized. Do you provide glycerol stocks of plasmids for SMARTchoice shrna constructs? Should I purchase an individual SMARTchoice shrna or the Set of 3? Could I pool my three constructs or test them separately? Is there a guarantee on the SMARTchoice shrna products? Do you provide custom SMARTchoice shrna constructs targeting species other than human, mouse and rat? How are the SMARTchoice shrna sequence designs different from those found in sigenome? No. The SMARTvector product line is only available as pre-packaged, ready-to-use lentiviral particles. During the discovery phase of your experiment you should test several different constructs to identify the one(s) with the greatest functionality in your cell system. For this reason, we recommend the Set of 3. The SMARTchoice shrna viral particles are intended to be tested as individuals for functionality. We do not recommend pooling multiple constructs. We have every expectation that the SMARTchoice shrna products will lead to high levels of silencing of the intended gene target; however, because of the individual biological differences in each cell system being used we cannot guarantee a specific level of silencing. We typically observe significant silencing from at least one out of three constructs for each gene. No. However, we do have three SMARTchoice shrna constructs pre-designed for every gene in human, mouse and rat. We have developed an algorithm specific for the highly processed microrna scaffold in which the SMARTchoice shrna targeting sequence of interest is incorporated. The algorithm was derived using the same successful bioinformatics strategies to build our rational design method, SMARTselection Design (Reynolds 1 ; Birmingham 2 ), and adapted for shrna designs as expressed from the SMARTvector microrna scaffold. Briefly, weighted criteria for features associated with silencing functionality were identified from a teaching set of functional and non-functional shrna sequences and combined into a selection algorithm. Of note, side-by-side comparisons of the top scoring sequences identified by the two rational design algorithms ( and shrna) show no overlap. This suggests that the functionality criteria for and shrna are not equivalent - an important factor when considering the design of shrna-based experimentation. 1. Reynolds, A. et al. (24) Rational Design for RNA Interference. Nature Biotechnology 22(3): Birmingham, A. et al. (27) A protocol for designing s with high functionality and specificity. Nature Protocols 2(9): Continued on next page. 12 Appendix Appendix 13

54 Dharmacon shrna products continued. Dharmacon microrna products QUESTIONS ANSWERS QUESTIONS ANSWERS What titers are obtained for the products supplied as lentiviral particles? How do I determine transduction efficiency when I use lentiviral particles with a chosen cell line? For viral particle preparations, which are then purified and concentrated, individual GIPZ or SMARTchoice shrnas, shmimic Lentiviral micrornas, and Precision LentiORFs, we are able to achieve titers of at least TU/mL, and Inducible SMARTchoice which achieves titers of TU/mL, as determined by quantification of TurboGFP expression in transduced HEK293T cells. Transduction efficiency can be measured by assessing the fraction of cells expressing TurboGFP or TurboRFP after transduction with lentiviral particles. This can be achieved by simple microscopic observation using a fluorescence microscope or by FACS analysis. Why are there multiple mimics available for my microrna of interest? We have designed miridian microrna Mimics and Hairpin Inhibitors for each mature microrna associated with a particular precursor microrna. Some mature microrna sequences can arise from multiple unique precursor sequences which results in several miridian products for the same mature sequence. The typical nomenclature for precursors giving rise to the same mature sequence is -1, -2,...-X following the microrna designation (e.g., hsa-mir and hsa-mir-218-2). In most cases, as long as the mature sequence is the same, the miridian products will be interchangable. You can download the sequences of the human mature micrornas used to design the shmimic micrornas on the mirbase database website, release 19.: ftp://mirbase.org/pub/mirbase/19./. How many cells can I transduce with the amount of lentiviral particles provided? How do I know if my target cell type can be transduced with Dharmacon lentiviral particles? What quality control is performed for monitoring lentiviral particle production? Are lentiviral particles safe to use in the laboratory? What precautions should be taken? Can lentiviral particles be further propagated in the lab? What is the shelf-life of the viral particle products? Can I use Virapower to package my lentiviral shrna construct? The number of cells that can be transduced will depend upon the MOIs used during the transduction procedure and the amount of virus you order (e.g., 5 μl or 1 μl). Procedures and formulae for optimizing transduction for your particular cell type are provided in the product-specific technical manuals for lentiviral particle products. We recommend you perform the optimization matrix using control viral particles, which is more cost-effective than using gene-specific experimental products to optimize. Lentiviral particles are pseudo-typed with the VSVg envelope protein to allow broad tropism across many human cells. To determine if your particular cell type is permissive to lentiviral transduction, please consult PubMed ( and other literature databases to ascertain whether others using your cell type have successfully performed lentiviral transductions. If a scan of the literature fails to provide any insights, we recommend assessing transduction using the SMARTchoice Promoter Selection Plate. Lentiviral particle products undergo stringent quality control at multiple steps during the manufacturing process. These include: Restriction enzyme analysis of the vector and sequencing of the entire insert to verify sequence integrity Viral titer confirmation by counting of TurboGFP-positive cells following transduction in HEK293T cells Examination of cells transduced with each batch of viral particles to ensure preparations are free from mold and bacterial contamination Lentiviral delivery vehicles have been employed in many research laboratories around the world without incident. Handling of lentiviral particles requires attention to standard BSL-2 guidelines. Details of the BSL-2 equipment, facilities and protocols can be found at rac/guidelines/appendix_g.htm. No. Dharmacon lentiviral constructs are engineered for maximum biosafety and are provided as replication-incompetent lentiviral particles. Dharmacon lentiviral particles can be stored at -8 C for 12 months without a significant loss in titer if left unthawed. Each thaw cycle can potentially reduce titer significantly, so multiple freezethaws should be avoided as much as possible. Virapower is a 3 rd generation lentiviral packaging system. It will NOT work for any of our second generation lentiviral vectors, including pgipz, ptripz and ploc because they have wild-type 5' LTRs that require the expression of the tat gene (which Virapower does not express) in the packaging system. Please use the Dharmacon Trans-Lentiviral shrna Packaging Kit to package GIPZ or TRIPZ constructs. Virapower would work for TRC shrna in the plko.1 vector because the plko.1 vector has a chimeric 5' LTR. However, the RNAi Consortium recommends the use of 2 nd generation packaging systems for increased viral titer. What is a microrna star strand? What is the best method to use for delivering miridian microrna Mimics and Hairpin Inhibitors into my cells? How long does inhibition last after the transfection of miridian microrna Hairpin Inhibitors? How do I assess functionality and performance for my microrna Mimics and Hairpin Inhibitors? Precursor micrornas often give rise to two strands, one 5 and one 3 to the precursor microrna hairpin loop. Historically, the most abundant strand was designated the mature strand and the less abundant strand was designated the *strand (star strand). Because the star symbol is not easily read and interpreted by computer programs, this nomenclature has been retired in favor of a 5 (-5p) and 3 (-3p) designation (More details in For example, hsa-mir-214-5p and hsa-mir-214-3p, used to be designated hsa-mir-214* and hsa-mir-214, respectively. The 5p and 3p strands can be easily distinguished by the mature sequence. Design features of both the miridian Mimics and Hairpin Inhibitors allow you to study the function of each mature microrna separately. The double-stranded Mimics have been modified to preferentially load the mature strand into RISC, while the Hairpin Inhibitors are designed to potently bind and inhibit the mature strand of the endogenous microrna. The miridian microrna Mimics and Hairpin Inhibitors can be delivered using standard lipid-mediated or electroporation delivery methods. The optimal methods and conditions for delivery will depend on the specific cell line or cell type that you are using. Conditions should be similar to those used for the delivery of other small nucleic acids (for example single-stranded oligos or s) into the same cell line. DharmaFECT Transfection Reagents can be used to transfect mimics and inhibitors. Cell line-specific transfection conditions are available in the DharmaFECT General Transfection protocol. The duration of inhibition is highly dependent upon the relative levels of expression of a particular microrna in the cell type being tested. Through a proprietary design and chemical modification pattern, miridian Hairpin Inhibitors are generally longer lasting and more durable than other inhibitor designs. Inhibition is typically observed for days, or even weeks, after transfection. We would suggest a timecourse experiment to determine empirically how long they will remain potent in your system. Many researchers use a reporter system to assess the change in microrna activity in response to the miridian Mimic or Hairpin Inhibitor treatment. The reporter construct can contain a completely complementary sequence to the mature microrna being studied (as a control), a putative microrna binding site or an entire 3' UTR of a putative target gene. In some cases, monitoring the expression of a known or putative target gene can be used for assessing microrna activity. micrornas can also be detected by RT-qPCR in much the same way that mrnas are detected. One factor to keep in mind is that you will need to add a polya tail for the RT step if you want to use oligo dt primers. There are several commercial vendors offering products for measuring microrna expression by RT-qPCR. Please make sure to consult the manufacturer's protocol for usage guidelines. For more information on this topic please see our Technical Note: Using Mimics and Inhibitors for microrna Functional Analysis. Continued on next page. 14 Appendix Appendix 15

55 Dharmacon microrna products continued. Dharmacon Custom Oligonucleotide Synthesis QUESTIONS What is the difference between a shmimic microrna and a synthetic microrna mimic? Is target gene downregulation by the shmimic Lentiviral micrornas transient or permanent? How do the shmimic microrna sequence designs differ from those in the synthetic miridian Mimics? ANSWERS shmimic Lentiviral micrornas are sold as lentiviral particles. When used to transduce a target cell line, the mature microrna will be expressed. Lentiviral delivery methods permit long-term expression of the mature microrna as well as providing the ability to target more biologically relevant cell lines that may be difficult-to-transfect. shmimic micrornas, by way of lentiviral particle delivery of the engineered vector, integrate their payload into the host cell genome. When cells are maintained under puromycin selection and as they divide, each recombinant genome is replicated and transferred to the daughter cell progeny and can permanently express the mature microrna. Thus, expression of the mature microrna is stable and permanent in these cells. However, microrna biology is complex and long-term down-regulation of putative gene targets depends on whether or not there is redundancy or involvement in feedback loops. Also, cell-dependent epigenetic regulatory mechanisms should be taken into account. The mature micrornas expressed from shmimic Lentiviral micrornas are the same as those represented in synthetic miridian Mimics. Thus, you can use both miridian shmimic microrna and synthetic Mimics with the assurances that the desired effector sequence that enters into RISC (the single-stranded, mature microrna) is correct and the primary microrna targeting element (the seed sequence) is preserved. QUESTIONS What are your capabilities for RNA synthesis? What is 2'-ACE chemistry and how does it compare to other RNA synthesis chemistries on the market? ANSWERS Dharmacon proprietary 2'-ACE chemistry allows for the synthesis of premium quality RNA oligos, even at lengths exceeding 8 nt. Synthesis yields are among the highest in the industry, with available synthesis scales in the nanomole to millimole range. Multiple options exist for chemical modification and postsynthesis processing. Please visit our website for full details. RNA applications can be executed with greater ease and confidence by utilizing the powerful advantages of the Dharmacon 2'-ACE technology. Greater reproducibility than any other chemistry Crude yields of unmatched purity without additional purification time and expense Maximum sequence fidelity and integrity due to enhanced coupling efficiency Download the Technical Note: 2'-ACE RNA Synthesis Chemistry for more information on the details of the Dharmacon RNA synthesis platform. All synthetic RNA oligos must be chemically protected at multiple sites during synthesis, regardless of the chemistry platform used. However, Dharmacon patented 2'-ACE synthesis chemistry allows for the quickest and mildest deprotection conditions compared to other chemistries like 2'-tBDMS and 2'-TOM. Short deprotection times and a mild chemical environment promote the highest level of purity for synthetic RNA in the marketplace today. When using shmimic micrornas, what controls should be included as part of a well-designed microrna modulation experiment? Will the human shmimic micrornas work in mouse and rat cell lines as well? We advise that both positive- and negative-control lentiviral treatments be assessed alongside untreated (no transduction) conditions in each microrna over-expression experiment, with each condition represented in triplicate, preferably in separate plates (if using multi-well plates). Using the highly functional human SMARTchoice shrna positive GAPD and validated Non-targeting or Empty Vector Control Particles will provide clean assessments of optimal transduction conditions. Finally, we recommend that shmimic microrna controls (shmimic negative controls and positive controls) be used to measure subtle effects attributable to microrna activity. The human miridian shmimic Lentiviral micrornas are designed using human mature microrna sequences from the mirbase database ( Many human micrornas are very well conserved across mammalian species. Where micrornas are conserved between human, mouse and rat, those shmimic micrornas could potentially be used in mouse and rat experiments. We recommend using the SMARTchoice Promoter Selection Plate to identify the optimal promoter for your specific cell of interest. Please contact our Technical Support team to get general recommendations for lentiviral transductions in rodent models. Do you synthesize DNA oligonucleotides? What chemical modifications are available for and single-stranded RNA? I can't find the chemical modification I need. Does this mean it is not available? Yes, we can synthesize DNA oligonucleotides. However, due to our experience and expertise in RNA synthesis, most of the DNA synthesis we do involves the generation of individual RNA/DNA chimeric molecules or special projects at a larger scale. Please inquire with our Technical Support team for special requests. We offer an extensive portfolio of chemical modifications for custom and single-stranded RNA. For, you can request a proprietary modification pattern be applied to your to enhance specificity (ON-TARGETplus), nuclease-resistance (sistable) or reagent-free cellular delivery (Accell). Stabilized microrna Mimics and Accell microrna Inhibitors are also available. Both and single-stranded RNA can be further manipulated with chemical modifications such as fluorescent dyes, linkers, biotin, cholesterol, thiol, and more. For a comprehensive list of modifications which can be ordered online, please visit our website. No. If the chemical modification you are looking for is not listed on our website, please inquire with our Technical Support team to verify availability. If it is not something we currently have in stock and are not able to obtain it commercially, we may even be able to make it for you as a custom chemistry service. Can you make custom RNA phosphoramidites? Yes, we regularly accept requests for synthesizing custom phosphoramidites or converting commercially available amidites to our 2'-ACE synthesis chemistry for incorporation into an RNA oligo. Please inquire with our Technical Support team for a feasibility determination and price quote. What are the different processing options I can choose from for custom synthesized RNA oligos? We offer multiple options for post-synthesis processing to meet varying individual needs. Both PAGE and HPLC are available for purification, as well as in vivo processing for experimentation with animal models. Please visit our website for a description of processing options. What is the difference between synthesis scale and final yield? The synthesis scale refers to the size of the RNA synthesis column; the final yield from any synthesis will vary depending on oligo length, sequence, modification(s). The final yield will be reduced further by any post-synthesis processing. We make every effort to provide the highest quality and the best yields possible. If you need a guaranteed final yield for any custom RNA or oligo please contact our Technical Support team for more information or to request a price quote. 16 Appendix Appendix 17

56 Dharmacon Gene Expression products Trademarks and license information QUESTIONS What makes an IMAGE cdna clone full-length? What is the rationale for offering viral particles for ORFs without a stop codon? What cdna or ORF clone verification methods should take place before and after purchase? Why is full-insert sequencing important? ANSWERS The IMAGE consortium uses a computer algorithm to analyze the full sequence of a cdna insert to make the determination of whether or not an IMAGE clone is full-length. Only clones determined to contain a complete coding sequence (CDS) are included in the 'full-length' category. We guarantee that the clone will contain the sequence that is listed in the associated accession. We strongly recommend that researchers review the sequence in the associated accession to determine if a clone will meet their needs. Suggestions for reviewing clone sequences can be found here: gelifesciences.com/dharmacon. NCBI's BLAST website offers a program for you to align two sequences - this allows you to examine the cdna clone sequence against your gene's full sequence: If you need a clone with a particular sequence, we recommend doing a BLAST search of our databases. All of our LentiORFs have a STOP codon, but the difference is the location. What we refer to as no stop means that the native stop codon of the ORF has been removed and instead of terminating at the last amino acid of that ORF, the translation of the protein goes on through the attb2 site and terminates immediately after that in the vector. This is done so that a LentiORF clone purchased as a glycerol stock can be modified by inserting a c-terminal fusion tag. The stop codon in the vector is flanked by unique sites so a customer can cut it out and replace with the tag of the choice. The trade off for this flexibility is ~13-15 extra amino acids at the C-terminus of the protein. When a Precision LentiORF clone is purchased as ready to use virus, it cannot be modified to make a fusion. Whether the LentiORF expression clone is stop or no stop is defined by the source ORF Entry clone from the ORFeome collaboration. The plate-row-column coordinates, DNA sequences and annotations of our cdna and ORF clones originate from the supplier and have not been independently verified for many of the collections. We do, however, curate and act upon customer-reported problems with clones. We therefore strongly recommend the following routine precautions: Prior to purchase, the customer should analyze the GenBank sequence for the clone of interest using BLAST or other bioinformatics tools, to ensure the sequence is appropriate for the planned experiment. After purchase, the customer should end-sequence the clone and align the result to the GenBank sequence to verify clone identity. EST and putative full-length clones may not be full-length because of incomplete ORFs, chimeric sequences or frameshifts. The entire sequence is needed for correct identification of alternative splice isoform cdnas that typically code for alternative proteins with alternative functions. Trademarks 214 General Electric Company. All rights reserved. alamarblue is a trademark of Accumed International, Inc. Alexa Fluor, Gateway, Lipofectamine 2, SuperScript, SYBR are registered trademarks of Life Technologies. ExGen is a trademark of Euromedex. Fugene is a registered trademark of Fugent LLC. HiPerfect is a trademark of QIAGEN Inc. QuantiGene is a trademark of Bayer. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Tet-On and Tet-Off is a registered trademark of CLONTECH Laboratories, Inc. TurboGFP and TurboRFP are trademarks of Evrogen. Solaris, TurboFect, ArrayScan, Verso and Maxima are trademarks of Thermo Fisher Scientific. CellTiter-Glo is a trademark of Promega Corporation. License information The Products, use and applications, are covered by pending and issued patents. It is each Purchaser s responsibility to determine if patent or other intellectual property rights held by third parties may restrict the use of Biological Materials for a particular application. Please refer to the relevant pages on gelifesciences.com/dharmacon 18 Appendix Appendix 19

57 Glossary Index PHRASE Antisense strand Complementary DNA (cdna) Clone Coding sequence (CDS) Dicer Drosha Exon Expressed Sequence Tag (EST) Gateway adapted Knockdown Knock out mirna/microrna Multiplicity of infection (MOI) Open Reading Frame (ORF) Phenotype RefSeq RNA Induced Silencing Complex (RISC) RNA interference (RNAi) Sense strand short hairpin RNA (shrna) small interfering RNA () EXPLANATION This represents the strand of an (or processed shrna) that is complementary to the target mrna; also referred to as the catalytic, targeting or guide strand. DNA synthesized from a mature mrna template in a reaction catalyzed by the enzyme Reverse Transcriptase. cdna is used to clone eukaryotic genes for exogenous expression in prokaryotes or in other eukaryotic cells and for amplification in RT-PCR and RT-qPCR. An exact copy of all or part of a macromolecule, for example DNA. Section of a gene or mrna that codes for a protein. A member of the RNase III family of nucleases present in the cytoplasm. It is involved in processing long dsrna and microrna into silencing intermediates that can interact with the RISC. A member of RNase III family of nucleases present in the nuclear compartment of a cell. It is the nuclease component of the Microprocessor complex and is responsible for the processing of naturally expressed primary (pri-) microrna transcripts into precursor (pre-) microrna hairpin structures. The products are then transported to the cytoplasm where they enter the RNAi pathway to regulate genes. Segments of genomic DNA which will be incorporated into the final mature mrna. Exons may include coding sequence, 5' untranslated region or the 3' untranslated region. A partial sequence (~5-8 bp) derived from a cdna. The data for the sequence information is limited typically to 3' or 5' end sequence. The EST is produced by one-shot sequencing of a cloned mrna. A cloning system that enables efficient transfer of DNA fragments between plasmids using a proprietary set of recombination sequences. It has effectively replaced the use of restriction endonucleases and ligases for some cloning applications. This process is patented by Life Technologies (formerly Invitrogen). Reverse genetic technique for reducing or silencing the target mrna or protein level as a result of RNAi. Classical genetic technique for deleting a gene from the genome of an organism to assess the gene function. Short, ~17-25 mer endogenously expressed RNA molecules that function as gene expression modulators. They are transcribed from non-coding sequences in the nucleus, processed and transported to the cytoplasm where they interact with the RISC to effect gene modulation. The ratio of transducing viral particles to cells. An MOI of 1 indicates that there are ten transducing units (TU) for every cell in the well. It is important to note that different cell types require different MOIs for successful transduction and knockdown of the target gene. The portion of an mrna that contains a sequence encoding a protein. The 5' untranslated region and 3' untranslated region are typically included upstream and downstream of this sequence. Observable features of cells or organisms, for example, shape, size, differentiation state, expression patterns. Curated database of gene sequences that represents non-redundant, annotated consensus or reference sequences derived from entries to the International Nucleotide Sequence Database Collaboration (INSDC). This is the sequence database used by the Dharmacon research team to design silencing intermediates for RNAi ( RefSeq/). This multi-component complex of proteins incorprates short RNA strands from microrna, or shrna to form the activated RISC. Activated RISC surveys the mrna population to identify its target and to effect sequence-specific gene silencing. A cellular mechanism by which silencing intermediates ( and microrna) reduce the expression of a target gene through a sequence-specific mechanism. The strand of an that is identical to the target mrna region; also referred to as the passenger or non-catalytic strand. Small RNA sequences or transcripts consisting of 19 to 29 base pair stems bridged by 4 to 9 nucleotide loops. In the cell, it is cleaved by Dicer-containing complex into resulting in target gene silencing or knockdown. A double-stranded RNA comprised of a 19 nucleotide core sequence with two nucleotide overhangs on the 3' end of each strand. One strand of the duplex is taken up by the RISC whereby the activated complex finds the corresponding message to silence or knockdown. By term 2 -ACE chemistry... 1, 48, 17 5X Buffer... 55, 64 Accell...22, 23, 25, 5, 55, 56, 64, 68, 12 Accell Delivery Media...23, 55, 64, 12 antisense-strands...2 apoptosis...1, 69, 71 BLAST... 18, 24, 49, 87, 18 branched DNA...24, 44, 6, 65, 7 cdna... 2, 3, 8, 42, 78, 79-85, 88, 93, 94, 99, 18, 11 C. elegans...8, 93, 94, 98 C. elegans ORFs...98 C. elegans transcription factors...98 cell cycle regulation... 69, 71 cell viability... 6, 64, 65, 7 Cherry-pick... 17, 2, 22, 24, 26, 27, 44, 45, 68, 74 cloning...33, 63, 78, 79, 85, 87, 98, 11 CMV promoter...32, 33, 42, 82, 85 codon... 82, 83, 84, 85, 87, 88, 18 custom RNA synthesis...46 custom shrna libraries...66 custom libraries...66 custom synthesis... 46, 48 cytokine receptors... 69, 71 Decode Pooled Lentiviral shrna Libraries...73 deubiquitinating enzymes... 69, 71 DharmaFECT Cell Culture Reagent...64, 7, 71 DharmaFECT Duo Transfection Reagent...6 DharmaFECT Transfection Reagents... 59, 6, 61, 64, 65, 71, 12, 15 dicer... 8, 11, 12, 13, 11 difficult-to-transfect cells...2, 22, 43, 87 DNA damage response... 69, 71 drosha... 8, 13, 11 druggable genome...17, 68, 69, 72, 73 drug targets... 69, 72 dsrna... 8, 11 Duet Libraries... 67, 72 E. coli Keio Knockout Strains...97 E. coli Promoter...97 E. coli Tagged ORFs...97 electroporation...17, 22, 41, 12, 15 ELISA...9 endogenous microrna pathway...9, 31 EST...99 Expressed Sequence Tag expression profiling... 12, 19 expression-ready lentiviral ORFs...82 false negatives...19 false positives...2 full insert sequencing...79 gain-of-function... 12, 41, 44, 78 gateway-adapted ORF... 82, 83 gene expression...2, 3, 11-13, 17, 25, 33-37, 4, 42-44, 54, 67, 7, 77-79, 81-83, 91, 97, 18, 11 gene function...8, 11, 28, 73, 78, 93, 95, 97, 11 gene knockdown... 2, 1, 11, 21, 23, 35, 36, 63, 7 gene search...79 genome... 3, 8, 11, 13, 16-18, 2, 24, 29, 41, 44, 45, 63, 66, 67, 78, 79-87, 91, 93-97, 11, 16, 11 GIPZ Lentiviral shrna... 28, 35, 54, 55 G Protein-Coupled Receptors (GPCR)...69, 71, 72 guaranteed silencing...16, 17, 18, 29, 38 guide strand...1, 19, 2, 11 high-titer... 14, 28, 29, 32, 33, 35, 39, 41, 42, 67, 81, 82, 87 hit stratification...66, 72, 75 Human ORFeome... 82, 83, 84, 85, 86, 88 inducible...2, 11, 15, 28, 29, 34, 36, 55, 57, 95 inducible promoter... 11, 29, 36, 95 in vivo RNAi...11, 13, 17, 29, 47, 12 ion channels...69, 71, 72 labeling... 52, 86 lentiviral delivery...35, 41, 14, 16 lentiviral particles...14, 28, 29, 32, 33, 39, 42, 58, 59, 63, 73, 82, 86, 87, 91, 13, 14, 16 Lincode for lncrna...26 Lincode...26 loss-of-function analysis...97 Mammalian Gene Collection (MGC)... 79, 81, 82, 83 membrane trafficking... 69, 71 microrna...3, 5, 7, 8, 9, 11-13, 15, 18, 19, 26-28, 3-32, 35, 36, 4-46, 48, 51, 54, 55, 57-61, 64, 66, 72, 74, 75, 1 microrna expression...12, 42, 45, 15 microrna inhibitors...12, 13, 51, 52, 74, 17 microrna mimics...2, 12, 13, 44, 51, 55, 64, 74, 15, 17 mirbase...12, 27, 42, 44, 45, 51, 74, 15 miridian Hairpin Inhibitors... 45, 51, 15 miridian libraries...74 miridian microrna mimics... 44, 51, 55, 74, 15 miridian shmimic Lentiviral micrornas mirna... 13, 24, 11 modified... 1, 2, 47, 5 MOI... 3, 33, 34, 35, 36, 42, 43, 73, 87, 14, 11 Molecular Barcoded Yeast (MoBY) ORFs...96 multiplicity of infection (MOI)... 3, 87 nuclear receptors... 69, 71 off-target effects...2 ON-TARGETplus...17, 2, 21, 25, 26, 55, 56, 67, 72, 11 open reading frame (ORF)...8 optimization...7, 71, 73, 75, 12, 14 ORF...2, 3, 8, 49, 78-83, 85-88, 91-99, 18, 11 ORFeome , 18 over-expression... 2, 8, 12, 78, 8, 88, 16 passenger strand...44 phenotype...1, 11, 12, 21, 36, 42, 72-74, 78, 96 phosphatase... 13, 69, 71-74, 88 Pol II...11, 29, 34 Pol III... 11, 29 potency...2, 18-2, 24, 45 Precison LentiORF...87 pre-designed... 16, 22, 24, 26, 47, 48, 51, 66, proteases...69, 71, 72 protein kinases...69, 71, 72 qpcr...2, 3, 26, 35-37, 42, 43, 45, 15, 11 rationally designed...2, 8, 4, 11 RefSeq...19, 26, 49, 11, 11 reporter gene...29, 34, 41, 55, 81 retrovirus...11 Reverse Transfection... 64, 66, 67, 7 Reverse Transfection Format (RTF) Libraries... 67, 7 RISC...8, 1, 12, 13, 2, 24, 4, 42, 44, 45, 48, RNAi...2, 3, 7-17, 19, 22, 26, 27, 29-31, 35-38, 41, 46, 47, 52, 54, 55, 57-59, 66, 67, 71, 73, 75, 78, 93, 94, 98, 11-14, 11 RNAi controls...7, 36, 53, 54, 71 RNAi Global Initiative...3, 75 RNAi libraries... 4, 5, 16, 27, 66 RNA interference (RNAi)...8, 13, 19, 28, 42, 52, 13, 11 RNA Polymerase...11, 37, 85 RNase-Free Water... 64, 74 RT-qPCR...42, 43, 45, 15, 11 seed region...8, 1, 12, 17, 18, 19, 2, 48, 11 selectable marker...11 sense-strand...2 serine proteases... 69, 71 short hairpin RNA...8, 13, 11 shrna...2, 3, 8, 11-15, 28-33, 35-39, 57-59, 63, 66-69, 73, 75, 81, 13, 14, 16, 11 sidesign Center... 4, 47, 49, 12 sigenome... 4, 24, 25, 55, 56, , 3, 8, 1-26, 46-5, 52, 54, 56, 58-61, 64-68, 7-72, 74, 75, 11-13, 15, 17, 11 sistable... 5, 55 SMARTchoice Inducible Lentiviral shrna... 34, 57 SMARTchoice shmimic... 41, 42 SMARTchoice shrna...11, 29, 32, 33, 34, 13, 14, 16 SMARTpool reagent...2, 24, 72 SMARTselection... 1, 18, 19, 26, 49, 12, 13 specificity...2, 1-13, 16-2, 22, 28, 3, 38, 41, 47, 48, 11, 13, 17 stable cell lines... 29, 41 Transduction The transfer of viral, bacterial or both bacterial and viral DNA from one cell to another using a viral vector. Continued on next page. 11 Appendix Appendix 111

58 synthetic RNA... 8, 53, 64, 17 target validation...8, 1, 72, 75 Tet-Promoter...95 tetracycline response element (TRE)...36 The ORFeome Collaboration Collection (OCC)...86 transcription factors... 69, 98 transduction..11, 28-3, 32, 33, 35-39, 41-43, 54, 57, 63, 67, 73, 81, 86, 87, 13, 14, 16, 11 transfection...2, 3, 8, 11, 12, 16, 17, 19, 22-25, 35-38, 41, 43-45, 47, 48, 51, 54-56, 58-65, 67, 7, 71, 83, 85-91, 11-13, 15 transfection indicators... 22, 54, 55, 57 Trans-Lentiviral shrna Packaging Kit... 59, 63, 14 TRC lentiviral shrna... 37, 55, 57, 68 TRE... 29, 36 TRIPZ Inducible Lentiviral shrna...36, 55, 57 TurboFect Transfection Reagent...37, 38, 59, 62, 89 TurboGFP... 29, 32, 34, 35, 41-43, 81, 87, 14 TurboRFP...29, 34, 36, 14 Tyrosine Kinases... 69, 71 ubiquitin conjugation...69, 71, 72 viral packaging...58 Western blot analysis... 22, 87 Xenopus Gene Collection (XGC)...99 Yeast Knockout Strains...95 Yeast Magic Marker Strains...95 Zebrafish cdna and ORF collections...99 By product name Accell Ancillary Reagents C. elegans Products CCSB-Broad Lentiviral Expression Library CCSB Human ORFeome Collection Custom microrna Modulation Tools Custom Single-stranded RNA Synthesis Custom Synthesis Decode Pooled Lentiviral shrna Libraries DharmaFECT Transfection Reagents... 6 Duet Libraries E.coli Products GIPZ Lentiviral shrna GIPZ & TRIPZ shrna Starter Kits Human ORFeome V Mammalian Gene Collection (MGC) microrna Controls microrna Libraries miridian microrna Hairpin Inhibitors miridian microrna Mimics SMARTchoice shmimic Lentiviral microrna Molecular Barcoded Yeast (MoBY) ORFs ON-TARGETplus Precision LentiORF Collection Pre-defined Gene Family and Pathway Libraries Lincode for lncrna Pre-defined Gene Family cdna and ORF Libraries.. 88 Reverse Transfection Format (RTF) Libraries... 7 shrna Controls sidesign Center sigenome Controls Reagents for in vivo Applications... 5 SMARTchoice shrna SMARTchoice Inducible Lentiviral shrna The ORFeome Collaboration Collection (OCC) Trans-Lentiviral ORF Packaging Kit Trans-Lentiviral shrna Packaging Kit TRC Lentiviral shrna TRIPZ Inducible Lentiviral shrna TurboFect in vivo Transfection Reagent... 9 TurboFect Transfection Reagent...62, 89 Xenopus Gene Collection (XGC) Appendix

59 For more information visit: gelifesciences.com/dharmacon 214 General Electric Company. All rights reserved.