TruSeq Stranded mrna. Reference Guide. For Research Use Only. Not for use in diagnostic procedures. Document # v00 October 2017

Transcription

1 TrSeq Stranded mrna Reference Gide Docment # v00 October 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 TrSeq Stranded mrna Reference Gide This docment and its contents are proprietary to Illmina, Inc. and its affiliates ("Illmina"), and are intended solely for the contractal se of its cstomer in connection with the se of the prodct(s) described herein and for no other prpose. This docment and its contents shall not be sed or distribted for any other prpose and/or otherwise commnicated, disclosed, or reprodced in any way whatsoever withot the prior written consent of Illmina. Illmina does not convey any license nder its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this docment. The instrctions in this docment mst be strictly and explicitly followed by qalified and properly trained personnel in order to ensre the proper and safe se of the prodct(s) described herein. All of the contents of this docment mst be flly read and nderstood prior to sing sch prodct(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY, AND WILL VOID ANY WARRANTY APPLICABLE TO THE PRODUCT(S). ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illmina, Inc. All rights reserved. Illmina, and the streaming bases design are registered or pending trademarks of Illmina, Inc. and/or its affiliate(s) in the U.S. and/or other contries. All other names, logos, and other trademarks are the property of their respective owners. Limited Use Label License: This prodct and its se are the sbject of one or more issed and/or pending U.S. and foreign patent applications owned by Max Planck Gesellschaft, exclsively licensed to New England Biolabs, Inc. and sblicensed to Illmina, Inc. The prchase of this prodct from Illmina, Inc., its affiliates, or its athorized resellers and distribtors conveys to the byer the non-transferable right to se the prchased amont of the prodct and components of the prodct by the byer (whether the byer is an academic or for profit entity). The prchase of this prodct does not convey a license nder any claims in the foregoing patents or patent applications directed to prodcing the prodct. The byer cannot sell or otherwise transfer this prodct or its components to a third party or otherwise se this prodct for the following COMMERCIAL PURPOSES: (1) se of the prodct or its components in manfactring; or (2) se of the prodct or its components for therapetic or prophylactic prposes in hmans or animals. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. ii

3 Table of Contents Chapter 1 Overview 1 Introdction 1 Process 1 RNA Inpt Recommendations 4 Additional Resorces 6 Chapter 2 Protocol 7 Introdction 7 Tips and Techniqes 7 Library Prep Workflow 9 Prepare for Pooling 9 Prify and Fragment mrna 9 Synthesize First Strand cdna 12 Synthesize Second Strand cdna 14 Adenylate 3 Ends 16 Ligate Adapters 17 Enrich DNA Fragments 20 Check Libraries 22 Normalize and Pool Libraries 23 Appendix A Spporting Information 26 Introdction 26 Prodct Contents 26 Inline Control DNA 28 Consmables and Eqipment 29 Index Adapter Seqences 31 Acronyms 31 Appendix B Alternate Fragmentation Protocols 33 Introdction 33 Modify RNA Fragmentation Time 33 Technical Assistance 35 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. iii

4 Chapter 1 Overview Introdction 1 Process 1 RNA Inpt Recommendations 4 Additional Resorces 6 Introdction This protocol explains how to convert the mrna in a total RNA sample into a library of template molecles of known strand origin sing the reagents provided in an Illmina TrSeq Stranded mrna library prep workflow. The library is sitable for sbseqent clster generation and DNA seqencing. This library prep protocol offers: Strand information an RNA transcript Library captre of both coding RNA and mltiple forms of noncoding RNA that are polyadenylated Optimized workflows for processing low sample (LS) and high sample (HS) nmbers in parallel Inclsive components: Library Prep components inclde library prep reagents exclding index adapters. Index adapter components mst be prchased separately. For more information, see Spporting Information on page 26. The se of the inclded In-line Control DNA provided with this kit is optional and reqires a cstom analysis pipeline. If analysis is not available, omit them from the prep. The protocol is compatible with no indexing or a lower indexing pooling level. The libraries generated do not reqire PCR amplification to enable clster generation, althogh PCR is recommended in the standard protocol to meet the yield reqirements of most standard applications. Process The following workflow explains how the TrSeq Stranded mrna Library Prep assay works, how strandedness is achieved, and which read maps to which strand. Prify and Fragment mrna The Poly-A containing mrna molecles are prified sing poly-t oligo attached magnetic beads. Following prification, the mrna is fragmented into small pieces sing divalent cations nder elevated temperatre. Figre 1 Prifying and Fragmenting mrna Synthesize First Strand cdna Cleaved RNA fragments are copied into first strand cdna sing reverse transcriptase and random primers. Adding Actinomycin D to FSA (First Stand Synthesis Act D mix) prevents sprios DNA-dependent synthesis, while allowing RNA-dependent synthesis, improving strand specificity. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 1

5 TrSeq Stranded mrna Reference Gide Figre 2 Synthesizing First Strand cdna Synthesize Second Strand cdna Strand specificity is achieved by replacing dttp with dutp in the SMM (Second Strand Marking Mix), followed by second strand cdna synthesis sing DNA Polymerase I and RNase H. The incorporation of dutp in second strand synthesis qenches the second strand dring amplification. Figre 3 Synthesizing Second Strand cdna Adenylate 3' Ends A single 'A' ncleotide is added to the 3' ends of the blnt fragments to prevent them from ligating to each other dring the adapter ligation reaction. A corresponding single 'T' ncleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensres a low rate of chimera (concatenated template) formation. Figre 4 Adenylating 3' Ends Ligate Adapters The single-index adapter is shown in this workflow. The dal-index adapter option is not shown in this workflow. Adapter ligation prepares the ds cdna for hybridization onto a flow cell. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 2

6 TrSeq Stranded mrna Reference Gide Figre 5 Ligating Adapters Enrich DNA Fragments Polymerase sed in the assay does not incorporate past dutp. Therefore, the second strand is effectively qenched dring amplification. The prodcts are enriched with PCR and prified to create the final cdna library. Figre 6 Enriching DNA Fragments Final Library The LS library featres a single-index adapter, as shown in this workflow. The HS library featres a dal-index adapter, which contains a niqe index at each end. The HS library dal-index adapter is not shown in this workflow. Figre 7 LS Final Library Clster Generation and Read 1 Seqencing In Read 1, seqencing reads map to the antisense strand. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 3

7 TrSeq Stranded mrna Reference Gide Figre 8 Clster Generation and Read 1 Seqencing Paired-end Trnarond and Read 2 Seqencing In Read 2, seqencing reads map to the sense strand. Figre 9 Paired-end Trnarond and Read 2 Seqencing RNA Inpt Recommendations Total RNA Inpt The protocol is optimized for µg of total RNA. Use a florometric method to qantify RNA. Lower amonts can reslt in inefficient ligation and low yield. The protocol has been tested sing µg of high-qality niversal hman reference total RNA as inpt. Use of RNA from other species, tisses, or lower qality RNA, inclding FFPE samples, might reqire frther optimization to determine the inpt amont. Dilte inline controls for tracking the conversion of dsdna into libraries. The diltion is optimized for µg of high-qality inpt RNA. When sing less RNA or RNA with very low mrna content, the controls might need frther diltion. If controls are not sed, se RSB (Resspension Bffer) in their place in the protocol. Determine the qality of the RNA starting material. The fragmentation conditions are optimized for high-qality RNA. Use an Agilent RNA 6000 Nano Kit or Advanced Analytical Standard Sensitivity RNA Analysis Kit to determine the qality of yor starting material. Do not se low qality or degraded RNA with this protocol. Use of degraded RNA can reslt in low yield, overrepresentation of the 3' ends of the RNA molecles, or failre of the protocol. Check total RNA integrity following isolation: On an Agilent Technologies 2100 Bioanalyzer, samples with an RNA Integrity Nmber (RIN) vale 8, or on an Advanced Analytical Fragment Analyzer, samples with an RNA Qality Nmber (RQN) vale 8, are recommended. Using RNA with DNA contamination reslts in an overestimation of the amont of RNA sed. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 4

8 TrSeq Stranded mrna Reference Gide Inclde a DNase step with the RNA isolation method to ensre prity and accrate qantification of the sample. The following figre shows a Universal Hman Reference (UHR) starting RNA Bioanalyzer trace. Figre 10 Starting RNA Bioanalyzer Trace Alternatively, rn a formaldehyde 1% agarose gel and determine the integrity of RNA pon staining with ethidim bromide. High-qality RNA shows a 28S ribosomal RNA (rrna) band at 4.5 kb with 2X the intensity of the 18S rrna band at 1.9 kb. Both kb determinations are relative to an RNA 6000 ladder. The mrna appears as a smear from kb. Prified mrna Inpt Yo can se ng previosly isolated mrna as starting material. Use the entire fraction of mrna prified from µg of total RNA. 1 Concentrate the mrna to 5 µl by ethanol precipitation or se a QIAGEN MinElte colmn before adding FPF (Fragment, Prime, Finish Mix). If ethanol precipitation is sed, resspend the pellet in 18 µl FPF. If a QIAGEN MinElte colmn is sed, elte the mrna with 5 µl moleclar biology-grade water and add 13 µl FPF. Using a MinElte colmn reslts in a loss of p to 50% of the mrna de to the low eltion volme. 2 Proceed to Fragment mrna on page 12 step 14. Positive Control Use Agilent Technologies Hman UHR total RNA (catalog # ) as a positive control sample for this protocol. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 5

9 TrSeq Stranded mrna Reference Gide Additional Resorces The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector TrSeq Stranded mrna Checklist (docment # xxxxx Index Adpater Pooling Gide (docment # ) Seqencing Library qpcr Qantification Gide (docment # ) Illmina Experiment Manager Gide (docment # ) and IEM TrSeq DNA, RNA, or ChIP Qick Reference Card (docment # ) BaseSpace help (help.basespace.illmina.com) Local Rn Manager Software Gide (docment # ) Description spport.illmina.com/cstom-protocol-selector.html A wizard for generating cstomized end-to-end docmentation that is tailored to the library prep method, rn parameters, and analysis method sed for the seqencing rn. Provides a checklist of the protocol steps. The checklist is intended for experienced sers. Provides pooling gidelines for preparing libraries for Illmina seqencing systems that reqire balanced index combinations. Review this gide before beginning library preparation. Describes a qpcr method for qantifying seqencing by synthesis (SBS) libraries generated sing the Illmina library prep protocols. Provide information abot creating and editing appropriate sample sheets for Illmina seqencing systems and analysis software and record parameters for yor sample plate. Provides information abot the BaseSpace seqencing data analysis tool that also enables yo to organize samples, libraries, pools, and seqencing rns in a single environment. Provides an overview of the Local Rn Manager (LRM) software, instrctions for sing software featres, and instrctions for installing analysis modles on the instrment compter. Visit the TrSeq Stranded mrna workflow spport page on the Illmina website for access to reqirements and compatibility, additional docmentation, software downloads, online training, freqently asked qestions, and best practices. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 6

10 Chapter 2 Protocol Introdction 7 Tips and Techniqes 7 Library Prep Workflow 9 Prepare for Pooling 9 Prify and Fragment mrna 9 Synthesize First Strand cdna 12 Synthesize Second Strand cdna 14 Adenylate 3 Ends 16 Ligate Adapters 17 Enrich DNA Fragments 20 Check Libraries 22 Normalize and Pool Libraries 23 Introdction Table 1 Perform the protocol in the order described sing specified volmes and incbation parameters. The protocol provides a single workflow with options depending on the nmber of samples processed. Differences for each option are designated with [HS] or [LS]. Follow the instrctions for the workflow option that spports yor nmber of samples. Yo can expect eqivalent reslts from either option. However, the [HS] option can yield more consistent reslts between samples. Each option incldes the following featres. Workflow Variable HS LS 48 sample workflow > 48 with index adapter tbes 48 with index adapter tbes 96 sample workflow > 24 with index adapter plate 24 with index adapter plate Plate Type Workflow Variations Incbation Eqipment 96-well Hard-Shell PCR plate 96-well midi plate 96-well thermal cycler Microheating system Mixing Method Microplate shaker Pipetting 96-well 0.3 ml PCR plate 96-well midi plate 96-well thermal cycler Review Best Practices before proceeding. See Additional Resorces on page 6 for information on how to access TrSeq Stranded mrna Library Prep Best Practices on the Illmina website. Before proceeding, confirm workflow contents and make sre that yo have the reqired eqipment and consmables. For more information, see Spporting Information on page 26. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Avoiding Cross-Contamination When adding or transferring samples, change tips between each sample. When adding adapters or primers, change tips between each row and each colmn. Remove nsed index adapter tbes from the working area. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 7

11 TrSeq Stranded mrna Reference Gide Sealing the Plate Always seal the 96-well plate before the following steps in the protocol: Shaking steps Vortexing steps Centrifge steps Thermal cycling steps Apply the adhesive seal to cover the plate, and seal with a rbber roller. Microseal 'B' adhesive seals are effective at -40 C to 110 C, and sitable for skirted or semiskirted PCR plates. Use Microseal 'B' for shaking, centrifging, and long-term storage. Microseal 'A' adhesive film is sed for thermal cycling steps to prevent evaporation. Plate Transfers When transferring volmes between plates, transfer the specified volme from each well of a plate to the corresponding well of the other plate. Centrifgation Centrifge at any step in the procedre to consolidate liqid or beads in the bottom of the well, and to prevent sample loss. Handling Beads Do not freeze beads. Pipette bead sspensions slowly. Before se, allow the beads to come to room temperatre. Immediately before se, vortex the beads ntil they are well dispersed. The color of the liqid mst appear homogeneos. Vortex throghot protocol as necessary to keep homogenos. If beads are aspirated into pipette tips, dispense back to the plate on the magnetic stand, and wait ntil the liqid is clear (~2 mintes). When washing beads: Use the specified magnetic stand for the plate. Dispense liqid so that beads on the side of the wells are wetted. Keep the plate on the magnetic stand ntil the instrctions specify to remove it. Do not agitate the plate while it is on the magnetic stand. Do not distrb the bead pellet. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 8

12 TrSeq Stranded mrna Reference Gide Library Prep Workflow Figre 11 TrSeq Stranded mrna Library Prep Workflow Prepare for Pooling When pooling samples for seqencing, se IEM, LRM, or BaseSpace Prep Tab to record information abot yor samples before beginning library preparation. Use IEM to create and edit sample sheets for Illmina seqencing systems and analysis software. Use LRM and BaseSpace Prep Tab to organize samples, libraries, pools, and a rn for Illmina seqencing systems and analysis software. Review the planning steps in the Index Adpater Pooling Gide (docment # ) and the Library pooling gidelines for the NextSeq and MiniSeq systems technical blletin when preparing libraries for Illmina seqencing systems that reqire balanced index combinations. Prify and Fragment mrna This process prifies the polya containing mrna molecles sing oligo-dt attached magnetic beads and 2 ronds of prification. Dring the second eltion of the polya RNA, the RNA is fragmented and primed for cdna synthesis. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 9

13 TrSeq Stranded mrna Reference Gide Consmables Total RNA samples (0.1 1 µg per sample) BBB (Bead Binding Bffer) BWB (Bead Washing Bffer) ELB (Eltion Bffer) FPF (Fragment, Prime, Finish Mix) RPB (RNA Prification Beads) RSB (Resspension Bffer) Barcode labels RBP (RNA Bead Plate) [HS] RFP (RNA Fragmentation Plate) Choose from the following containers: [HS] 96-well Hard-Shell 0.3 ml PCR plate (1) and 96-well midi plate (1) [LS] 96-well 0.3 ml PCR plate, semiskirted or skirtless Microseal 'B' adhesive seals Preparation 1 Prepare the following consmables. Item Storage Instrctions FPF -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. RSB -25 C to -15 C Thaw at room temperatre. Store at 2 C to 8 C after the initial thaw. BBB 2 C to 8 C Thaw at room temperatre. Retrn to storage after se. BWB 2 C to 8 C Thaw at room temperatre. Retrn to storage after se. ELB 2 C to 8 C Thaw at room temperatre. Retrn to storage after se. RPB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Save the following Eltion 2-Frag-Prime program on the thermal cycler. Choose the preheat lid option and set to 100 C 94 C for 8 mintes Hold at 4 C For inserts larger than bp with a median size of 150 bp, see Alternate Fragmentation Protocols on page [LS] Save the following mrna Denatration program on the thermal cycler. Choose the preheat lid option and set to 100 C 65 C for 5 mintes Hold at 4 C Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 10

14 TrSeq Stranded mrna Reference Gide 4 [LS] Save the following mrna Eltion 1 program on the thermal cycler. Choose the preheat lid option and set to 100 C 80 C for 2 mintes Hold at 25 C 5 [HS] Preheat the microheating system to 65 C. 6 Set the centrifge to 15 C to 25 C. 7 [HS] Calibrate the microplate shaker to 1000 rpm sing a stroboscope. 8 Apply barcode labels to plates as follows. RBP [midi or PCR plate] [HS] RFP [Hard-Shell PCR plate] Procedre Prify mrna 1 Dilte the total RNA in nclease-free ltrapre water to a final volme of 50 μl in each well of the RBP plate. 2 Vortex RPB ntil well-dispersed. 3 Add 50 µl RPB to each well, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 1 minte. [LS] Pipette p and down 6 times. 4 Incbate as follows. [HS] Place on the 65 C microheating system with the lid closed for 5 mintes, and then place on ice for 1 minte. [LS] Place on the thermal cycler and rn the mrna Denatration program. Each well contains 100 µl. 5 Seal the RBP plate with a Microseal 'B' adhesive seal before rnning the mrna denatration program. 6 Place on the bench and incbate at room temperatre for 5 mintes. 7 [HS] Preheat the microheating system to 80 C for sbseqent incbation. 8 [LS] Centrifge at 280 g for 1 minte. 9 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 10 Remove and discard all spernatant from each well. 11 Remove from the magnetic stand. 12 Add 200 µl BWB to each well, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 1 minte. [LS] Pipette p and down 6 times. 13 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 14 Remove and discard all spernatant from each well. 15 Remove from the magnetic stand. 16 Add 50 µl ELB to each well, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 1 minte. [LS] Pipette p and down 6 times. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 11

15 TrSeq Stranded mrna Reference Gide 17 [LS] Centrifge at 280 g for 1 minte. 18 Incbate as follows. [HS] Place on the 80 C microheating system with the lid closed for 2 mintes, and then place on ice for 1 minte. [LS] Place on the thermal cycler and rn the mrna Eltion 1 program. Each well contains 50 µl. 19 Place on the bench. Fragment mrna 1 Add 50 µl BBB to each well, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 1 minte. [LS] Pipette p and down 6 times. 2 Seal the RBP plate with a Microseal 'B' adhesive seal before rnning the mrna denatration program. 3 Incbate at room temperatre for 5 mintes. 4 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 5 Remove and discard all spernatant from each well. 6 Remove from the magnetic stand. 7 Add 200 µl BWB to each well, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 1 minte. [LS] Pipette p and down 6 times. 8 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 9 Remove and discard all spernatant from each well. 10 Remove from the magnetic stand. 11 Add 19.5 µl FPF to each well, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 1 minte. [LS] Pipette p and down. 12 [LS] Centrifge at 280 g for 1 minte. 13 [HS] Transfer all to the corresponding well of the RFP plate. 14 Place on the thermal cycler and rn the Eltion 2 - Frag - Prime program. Each well contains 19.5 µl. 15 Centrifge briefly. Synthesize First Strand cdna This process reverse transcribes the cleaved RNA fragments primed with random hexamers into first strand cdna. The addition of Actinomycin D to the FSA (First Strand Synthesis Act D Mix) prevents sprios DNAdependent synthesis, while allowing RNA-dependent synthesis, and improving strand specificity. Consmables FSA (First Strand Synthesis Act D Mix) CDP (cdna Plate) barcode label SperScript II Reverse Transcriptase or Protoscript II Reverse Transcriptase (Use part # for 50 reactions. Make sre to have a qantity of two when sing 96 samples) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 12

16 TrSeq Stranded mrna Reference Gide Choose from the following containers: [HS] 96-well Hard-Shell 0.3 ml PCR plate [LS] 96-well 0.3 ml PCR plate, semiskirted or skirtless Microseal 'B' adhesive seals WARNING FSA contains Actinomycin D, a toxin. Personal injry can occr throgh inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any nsed contents in accordance with the governmental safety standards for yor region. See the safety data sheet (SDS) for environmental, health, and safety information. For more information, see Technical Assistance on page 35. Preparation 1 Prepare the following consmables. Item Storage Instrctions FSA -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. 2 Save the following Synthesize 1st Strand program on the thermal cycler: Choose the preheat lid option and set to 100 C 25 C for 10 mintes 42 C for 15 mintes 70 C for 15 mintes Hold at 4 C 3 Apply the CDP barcode label to a Hard-Shell PCR or PCR plate. Procedre 1 Place the RBP plate on the magnetic stand and wait ntil the liqid is clear (~5 mintes). 2 Transfer 17 µl spernatant to the corresponding well of the CDP plate. 3 Centrifge FSA at 600 g for 5 seconds. 4 Add 50 µl SperScript II to one tbe of FSA. Pipette to mix, and then centrifge briefly. Label the FSA tbe to indicate that SperScript II has been added. NOTE If yo are not sing the entire contents of FSA, add SperScript II at a ratio of 1 µl SperScript II to 9 µl FSA. The mixtre can be sed for sbseqent experiments. For more than 6 freeze-thaw cycles, prepare 10 µl aliqots and store at -25 C to -15 C. 5 Add 8 µl FSA and SperScript II mixtre to each well of the DFP plate, and then mix thoroghly as follows. [HS] Shake at 1600 rpm for 20 seconds. [LS] Pipette p and down. 6 Centrifge at 280 g for 1 minte. 7 Place on the preprogrammed thermal cycler and rn the Synthesize 1st Strand program. Each well contains 25 µl. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 13

17 TrSeq Stranded mrna Reference Gide Synthesize Second Strand cdna This process removes the RNA template, synthesizes a replacement strand, and incorporates dutp in place of dttp to generate ds cdna. The incorporation of dutp qenches the second strand dring amplification. Magnetic beads separate the ds cdna from the second strand reaction mix. The reslt is blnt-ended cdna. Consmables RSB (Resspension Bffer) SMM (Second Strand Marking Master Mix) AMPre XP beads [Optional] CTE (End Repair Control) Barcode labels ALP (Adapter Ligation Plate) [HS] CCP (cdna Clean Up Plate) Freshly prepared 80% ethanol (EtOH) Choose from the following containers: [HS] 96-well midi plates (2) [LS] 96-well 0.3 ml PCR plate, semiskirted or skirtless Microseal 'B' adhesive seals Abot Reagents Using CTE is optional. Use eqal volme of RSB as a sbstitte. Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions CTE -25 C to -15 C Thaw at room temperatre, and then place on ice. SMM -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Choose the thermal cycler preheat lid option and set the lid to 30 C 3 Preheat the thermal cycler to 16 C. 4 Apply barcode labels to plates as follows. ALP [midi or PCR] [HS] CCP [midi] Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 14

18 TrSeq Stranded mrna Reference Gide Procedre Add SMM 1 Centrifge CTE at 600 g for 5 seconds. 2 Dilte CTE to 1:50 in RSB. For example, 2 μl CTE + 98 μl RSB. 3 Add 5 µl dilted CTE to each well. Discard dilted CTE after se. 4 Centrifge SMM at 600 g for 5 seconds. 5 Add 20 µl SMM to each well, and then mix thoroghly as follows. [HS] Shake at 1600 rpm for 20 seconds. [LS] Pipette p and down 6 times. 6 Centrifge at 280 g for 1 minte. 7 Place on the preprogrammed thermal cycler and incbate at 16 C for 1 hor. Each well contains 50 µl. 8 Place on the bench and let stand to bring to room temperatre. Prify cdna 1 [HS] Add AMPre XP beads as follows. a b Add 90 µl AMPre XP beads to the CCP plate. Transfer all from the CDP plate to the corresponding well of the CCP plate. 2 [LS] Add 90 µl AMPre XP beads to each well of the CDP plate. 3 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 4 Incbate at room temperatre for 15 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 7 Remove and discard 135 µl spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air-dry on the magnetic stand for 15 mintes. Do not over dry beads. 11 Remove from the magnetic stand. 12 Add 17.5 µl RSB to each well, and then mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 13 Incbate at room temperatre for 2 mintes. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 15

19 TrSeq Stranded mrna Reference Gide 14 Centrifge at 280 g for 1 minte. 15 Place on a magnetic stand and wait ntil the liqid is clear (~5 mintes). 16 Transfer 15 µl spernatant to the corresponding well of the ALP plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Adenylate 3 Ends One adenine (A) ncleotide is added to the 3ʹ ends of the blnt fragments to prevent them from ligating to each other dring adapter ligation reaction. One corresponding thymine (T) ncleotide on the 3ʹ end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensres a low rate of chimera (concatenated template) formation. Consmables ATL (A-Tailing Mix) RSB (Resspension Bffer) [Optional] CTA (A-Tailing Control) Microseal 'B' adhesive seals Abot Reagents Using CTA is optional. Use eqal volme of RSB as a sbstitte. Preparation 1 Prepare the following consmables. Item Storage Instrctions ATL -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. CTA -25 C to -15 C Thaw at room temperatre, and then place on ice. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 [HS] Preheat two microheating systems, one to 37 C and the other to 70 C. 3 [LS] Save the following ATAIL70 program on the thermal cycler: Choose the preheat lid option and set to 100 C 37 C for 30 mintes 70 C for 5 mintes Hold at 4 C Procedre 1 Centrifge CTA at 600 g for 5 seconds. 2 Dilte CTA to 1:100 in RSB. For example, 1 μl CTA + 99 μl RSB. 3 Add 2.5 µl dilted CTA to each well. Discard dilted CTA after se. 4 Centrifge ATL at 600 g for 5 seconds. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 16

20 TrSeq Stranded mrna Reference Gide 5 Add 12.5 µl ATL to each well, and then mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 6 Seal the ALP plate with a Mircoseal 'B' adhesive seal. 7 Centrifge at 280 g for 1 minte. 8 [HS] Incbate as follows. a b c Place on the 37 C microheating system with the lid closed for 30 mintes. Move to the 70 C microheating system with the lid closed for 5 mintes. Place on ice for 1 minte. 9 [LS] Incbate as follows. a b Place on the thermal cycler and rn the ATAIL70 program. Each well contains 30 µl. Centrifge at 280 g for 1 minte. Ligate Adapters This process ligates mltiple indexing adapters to the ends of the ds cdna fragments, which prepares them for hybridization onto a flow cell. Index adapters mst be ordered separately from the Library Prep components. For information on compatible index adapters, see Spporting Information on page 26. Consmables LIG (Ligation Mix) RNA Adapters (tbes or index adapter plate) RSB (Resspension Bffer) AMPre XP beads STL (Stop Ligation Bffer) [Optional] CTL (Ligation Control) Barcode labels CAP (Clean Up ALP Plate) PCR (Polymerase Chain Reaction Plate) [HS workflow] RAP (Index Adapter Plate) Freshly prepared 80% ethanol (EtOH) Choose from the following containers: [HS] 96-well midi plate and 96-well Hard-Shell 0.3 ml PCR plate [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2) Microseal 'B' adhesive seals Abot Reagents Using CTL is optional. Use an eqal volme of RSB as a sbstitte. Do not remove LIG from storage ntil instrcted to do so in the procedre. Retrn LIG to storage immediately after se. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 17

21 TrSeq Stranded mrna Reference Gide Vortex AMPre XP beads before each se. Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions CTL -25 C to -15 C Thaw at room temperatre, and then place on ice. RNA Adapters -25 C to -15 C Thaw at room temperatre for 10 mintes. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. STL -25 C to -15 C Thaw at room temperatre. Retrn to storage after se. 2 [HS] Preheat a microheating system to 30 C. 3 [LS] Save the following LIG program on the thermal cycler: Choose the preheat lid option and set to 100 C 30 C for 10 mintes Hold at 4 C 4 Label plates as follows. Apply a CAP barcode label to a midi or PCR plate. Apply a PCR barcode label to a Hard-Shell PCR or PCR plate. Procedre Add Index Adapters 1 [HS] Remove the tape seal from the appropriate Index Adapter Plate. 2 Centrifge the RNA Adapters as follows. Reagent Speed Dration Adapter tbes 600 g 5 seconds Index Adapter Plate 280 g 1 minte 3 [HS] Prepare the Index Adapter Plate as follows. a b Remove the plastic cover. Apply the Index Adapter Plate barcode label. 4 Centrifge CTL at 600 g for 5 seconds. 5 Dilte CTL 1:100 in RSB. For example, 1 μl CTL + 99 μl RSB. Discard the dilted CTL after se. 6 Remove LIG from -25 C to -15 C storage. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 18

22 TrSeq Stranded mrna Reference Gide 7 Add the following reagents in the order listed to each well. Dilted CTL (2.5 µl) LIG (2.5 µl) RNA adapters (2.5 µl) 8 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 9 Centrifge at 280 g for 1 minte. 10 [HS] Place on the 30 C microheating system with the lid closed for 10 mintes, and then place on ice. 11 [LS] Place on the thermal cycler and rn the LIG program. Each well contains 37.5 µl. 12 Centrifge STL at 600 g for 5 seconds. 13 Add 5 µl STL to each well, and then mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 14 Centrifge at 280 g for 1 minte. Clean Up Ligated Fragments 1 Perform steps 2 throgh 17 sing the Rond 1 volmes. 2 Add AMPre XP beads to each well. Rond 1 Rond 2 AMPre XP beads 42 µl 50 µl 3 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 4 Incbate at room temperatre for 15 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 7 Remove and discard all spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air-dry on the magnetic stand for 15 mintes. 11 Remove from the magnetic stand. 12 Add RSB to each well. Rond 1 Rond 2 RSB 52.5 µl 22.5 µl Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 19

23 TrSeq Stranded mrna Reference Gide 13 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down. 14 Incbate at room temperatre for 2 mintes. 15 Centrifge at 280 g for 1 minte. 16 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 17 Transfer 50 µl spernatant to the corresponding well of the CAP plate. 18 Repeat steps 2 throgh 17 with the new plate sing the Rond 2 volmes. 19 Transfer 20 µl spernatant to the corresponding well of the PCR plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Enrich DNA Fragments This process ses PCR to selectively enrich those DNA fragments that have adapter molecles on both ends and to amplify the amont of DNA in the library. PCR is performed with PPC (PCR Primer Cocktail) that anneals to the ends of the adapters. Minimize the nmber of PCR cycles to avoid skewing the representation of the library. NOTE Fragments with no adapters cannot hybridize to srface-bond primers in the flow cell. Fragments with an adapter on 1 end can hybridize to srface bond primers, bt cannot form clsters. Consmables PMM (PCR Master Mix) PPC (PCR Primer Cocktail) RSB (Resspension Bffer) AMPre XP beads TSP1 (Target Sample Plate) barcode label Freshly prepared 80% ethanol (EtOH) Choose from the following containers: [HS] 96-well Hard-Shell 0.3 ml PCR plate [LS] 96-well 0.3 ml PCR plate, semiskirted or skirtless Microseal 'A' film Microseal 'B' adhesive seals NOTE Use Microseal 'A' when sealing the plate before placing it on the thermal cycler. Use Microseal 'B' for other steps that reqire a sealed plate. Abot Reagents Vortex AMPre XP beads before each se. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 20

24 TrSeq Stranded mrna Reference Gide Vortex AMPre XP beads freqently to make sre that beads are evenly distribted. Aspirate and dispense AMPre XP beads slowly de to the viscosity of the soltion. Preparation 1 Prepare the following consmables. Item Storage Instrctions PPC -25 C to -15 C Thaw at room temperatre. Invert to mix, then centrifge at 600 g for 1 minte. Do not vortex. Retrn to storage after se. PMM -25 C to -15 C Thaw on ice. Invert to mix, then centrifge at 600 g for 1 minte. Do not vortex. Retrn to storage after se. RSB 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. AMPre XP beads 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Save the following mrna PCR program on the thermal cycler: Choose the preheat lid option and set to 100 C 98 C for 30 seconds 15 cycles of: 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds 72 C for 5 mintes Hold at 4 C 3 Apply the TSP1 barcode label to a Hard-Shell PCR or PCR plate. Procedre Amplify DNA Fragments 1 Place PCR plate on ice and add 5 μl PPC to each well. 2 Add 25 µl PMM to each well, and then mix thoroghly as follows. [HS] Shake at 1600 rpm for 20 seconds. [LS] Pipette p and down 10 times. 3 Centrifge at 280 g for 1 minte. 4 Place on the preprogrammed thermal cycler and rn the mrna PCR program. Each well contains 50 µl. Clean Up Amplified DNA 1 Centrifge at 280 g for 1 minte. 2 Add AMPre XP beads to each well. The volme depends on the type of adapter sed. Adapter Type Adapter tbes 50 µl Index Adapter Plate 47.5 µl Volme AMPre XP beads Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 21

25 TrSeq Stranded mrna Reference Gide 3 Mix thoroghly, as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 4 Incbate at room temperatre for 15 mintes. 5 Centrifge at 280 g for 1 minte. 6 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 7 Remove and discard all spernatant from each well. 8 Wash two times as follows. a b c Add 200 µl fresh 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 9 Use a 20 µl pipette to remove residal EtOH from each well. 10 Air-dry on the magnetic stand for 15 mintes. 11 Remove from the magnetic stand. 12 Add 32.5 µl RSB to each well, and then mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 13 Incbate at room temperatre for 2 mintes. 14 Centrifge at 280 g for 1 minte. 15 Place on a magnetic stand and wait ntil the liqid is clear (2 5 mintes). 16 Transfer 30 µl spernatant to the corresponding well of the TSP1 plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Check Libraries Qantify Libraries To achieve the highest-qality data on Illmina seqencing platforms, it is important to create optimm clster densities across every lane of the flow cell. Optimizing clster densities reqires accrate qantification of DNA libraries. 1 Qantify the libraries sing qpcr according to the llminaseqencing Library qpcr Qantification Gide (docment # ). Check Library Qality 1 If sing a Standard Sensitivity NGS Fragment Analysis Kit on an Advanced Analytical Fragment Analyzer: a b Dilte the DNA library 1:1 with RSB. Rn 1 µl dilted DNA library. 2 If sing a DNA 1000 chip on an Agilent Technologies 2100 Bioanalyzer, rn 1 µl ndilted DNA library. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 22

26 TrSeq Stranded mrna Reference Gide 3 Check the size and prity of the sample. Expect the final prodct to be a band at ~260 bp. Figre 12 Example Library Size Distribtion Figre 13 TrSeq Stranded mrna Library Prep 260 bp PCR Prodct Normalize and Pool Libraries This process describes how to prepare DNA templates for clster generation. Indexed DNA libraries are normalized to 10 nm in the DCT plate and then pooled in eqal volmes in the PDP plate. Non-indexed DNA libraries are normalized to 10 nm in the DCT plate. NOTE For best practice, perform normalization and pooling directly prior to seqencing. To minimize index hopping, do not store libraries in the pooled form. For more information, see Minimize index hopping in mltiplexed rns on the Illmina website. Consmables Barcode labels DCT (Dilted Clster Template) PDP (Pooled DCT Plate) (for pooling only) Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 23

27 TrSeq Stranded mrna Reference Gide Tris-HCl 10 mm, ph 8.5 with 0.1% Tween 20 Choose from the following containers: [HS] 96-well midi plate [HS] 96-well Hard-Shell 0.3 ml PCR plate (for pooling) [LS] 96-well midi plates (2) (second plate for pooling > 40 samples) [LS] 96-well 0.3 ml PCR plate, semiskirted or skirtless (for pooling 40 samples) Microseal 'B' adhesive seals Preparation 1 Apply barcode labels to plates as follows. DCT [midi plate] [For pooling only] PDP [Hard-Shell PCR or midi (> 40 samples) or PCR ( 40 samples) plate] Procedre Normalize Libraries 1 Transfer 10 µl library to the corresponding well of the DCT plate. 2 Normalize the library concentration with Tris-HCl 10 mm, ph 8.5 with 0.1% Tween 20 to 10 nm, and then mix thoroghly as follows. [HS] Shake at 1000 rpm for 2 mintes. [LS] Pipette p and down 10 times. NOTE Depending on the yield qantification data of each library, the final volme of each well can vary from µl. 3 Centrifge at 280 g for 1 minte. 4 Do the following, To pool libraries, proceed to the next step in the workflow. Libraries that are not pooled, mst be dilted and denatred before proceeding to clster generation. For more information, see the Dilte and Denatre gide for yor Illmina platform. Pool Libraries The pooling procedre depends on the nmber of libraries being pooled. Pool 2 24 Libraries 1 Transfer 10 µl of each normalized library to a single well of the PDP plate. 2 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 3 [HS] Centrifge at 280 g for 1 minte. 4 Proceed to clster generation. For more information, see the system gide for yor Illmina seqencing platform. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 24

28 TrSeq Stranded mrna Reference Gide Pool Libraries 1 Transfer 5 µl of each colmn of normalized library to colmn 1 of the PDP plate, and then mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 2 Centrifge at 280 g for 1 minte. 3 Transfer the contents of each well of colmn 1 to well A2. 4 Mix thoroghly as follows. [HS] Shake at 1800 rpm for 2 mintes. [LS] Pipette p and down 10 times. 5 Centrifge at 280 g for 1 minte. 6 Proceed to clster generation. For more information, see the system gide for yor Illmina seqencing platform. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to 7 days. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 25

29 Appendix A Spporting Information Spporting Information Introdction 26 Prodct Contents 26 Inline Control DNA 28 Consmables and Eqipment 29 Index Adapter Seqences 31 Acronyms 31 Introdction The protocol described in this gide assmes that yo have reviewed the contents of this section, confirmed workflow contents, and obtained all reqired consmables and eqipment. Prodct Contents Make sre that yo have all the reagents identified in this section before starting the protocol. The following library prep and index adapter components are available to order throgh Illmina to spport the TrSeq Stranded mrna library prep workflow. From Illmina, order one catalog nmber for the library prep component and one catalog nmber for the index adapter component depending on the nmber of samples for yor experiment. Library Prep Component Catalog # TrSeq Stranded mrna Library Prep (48 Samples) TrSeq Stranded mrna Library Prep (96 Samples) Index Adapter Component Catalog # IDT for Illmina-TrSeq RNA UD Indexes (24 indexes, 96 samples) IDT for Illmina-TrSeq RNA UD Indexes (96 indexes, 96 samples) TrSeq RNA Combinatorial Dal Indexes (96 indexes, 96 samples) TrSeq RNA Single Indexes (12 indexes, 24 samples) Set A TrSeq RNA Single Indexes (12 indexes, 24 samples) Set B TrSeq Stranded mrna Library Prep (48 Samples) This workflow ses the components described in the sections that follow. Library Prep Box 1, Store as specified Qantity Reagent Description Storage Temperatre 1 RPB RNA Prification Beads 2 C to 8 C 1 DTE CTE Diltion Tbe Room Temperatre 1 DTA CTA Diltion Tbe Room Temperatre 1 DTL CTL Diltion Tbe Room Temperatre Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 26

30 TrSeq Stranded mrna Reference Gide Library Prep Box 2, Store as specified This box also contains plate barcode labels. Qantity Reagent Description Storage Temperatre 1 BBB Bead Binding Bffer 2 C to 8 C 1 BWB Bead Washing Bffer 2 C to 8 C 1 ELB Eltion Bffer 2 C to 8 C 1 FPF Fragment, Prime, Finish Mix -25 C to -15 C Core Library Prep Box 1, Store at -25 C to -15 C Qantity Reagent Description 1 ATL A-Tailing Mix 1 CTA A-Tailing Control 1 CTE End Repair Control 1 CTL Ligation Control 1 LIG Ligation Mix 1 RSB Resspension Bffer 1 STL Stop Ligation Bffer Core Library Prep Box 2, Store at -25 C to -15 C Qantity Reagent Description 1 PMM PCR Master Mix 1 PPC PCR Primer Cocktail 1 FSA First Strand Synthesis Act D Mix 1 SMM Second Strand Marking Master Mix TrSeq Stranded mrna Library Prep (96 Samples) This workflow ses the components described in the sections that follow. A qantity of two of each box is inclded for the 96 sample workflow. Library Prep Box 1, Store as specified Qantity Reagent Description Storage Temperatre 2 RPB RNA Prification Beads 2 C to 8 C 1 DTL CTL Diltion Tbe Room Temperatre 1 DTE CTE Diltion Tbe Room Temperatre 1 DTA CTA Diltion Tbe Room Temperatre Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 27

31 TrSeq Stranded mrna Reference Gide Library Prep Box 2, Store as specified This box also contains plate barcode labels. Qantity Reagent Description Storage Temperatre 2 BBB Bead Binding Bffer 2 C to 8 C 2 ELB Eltion Bffer 2 C to 8 C 2 BWB Bead Washing Bffer 2 C to 8 C 2 FPF Fragment, Prime, Finish Mix -25 C to -15 C Core Library Prep Box 1, Store at -25 C to -15 C Qantity Reagent Description 1 ATL A-Tailing Mix 1 CTA A-Tailing Control 1 CTE End Repair Control 1 CTL Ligation Control 1 LIG Ligation Mix 1 RSB Resspension Bffer 1 STL Stop Ligation Bffer Core Library Prep Box 2, Store at -25 C to -15 C Qantity Reagent Description 2 PMM PCR Master Mix 2 PPC PCR Primer Cocktail 2 FSA First Strand Synthesis Act D Mix 2 SMM Second Strand Marking Master Mix Inline Control DNA The se of the inclded In-line Control DNA provided with this kit is optional and reqires a cstom analysis pipeline. If analysis is not available, Illmina recommends omitting them from the prep. CTE (End Repair Control), CTA (A-Tailing Control), and CTL (Ligation Control) contain DNA fragments sed as controls for the enzymatic activities of the SMM (Second Strand Marking Master Mix), ATL (A-Tailing Mix), and LIG (Ligation Mix). Each inline control contains dsdna fragments designed to report the sccess or failre of a specific enzymatic activity. The control molecles work throgh the design of their ends. Controls are added to the reactions before their corresponding step in the protocol. Their end strctres match the end strctres of a DNA molecle that has not gone throgh the step. If the step is sccessfl, the control molecle is modified to participate in downstream reactions of library generation and reslt in seqencing data. If the step fails, the control molecle does not go forward in the process and no seqencing data are generated. Using 1 µg of starting material, the controls yield approximately 0.2% of clsters, althogh the yield can vary based on library yield. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 28

32 TrSeq Stranded mrna Reference Gide Table 2 Reagent Fnction Control Strctre of Control DNA Ends SMM End repair: Generate blnt ended fragments by 3' > 5' exonclease and 5' > 3' polymerase activities SMM ATL LIG Inline Control Fnctions End repair: Add 5'-phosphate grops needed for downstream ligation A-tailing: Make fragments compatible with adapters and prevent self-ligation by adding a 3'-A overhang Ligation: Join 3'-T overhang adapters to 3'-A overhang inserts End Repair Control 1* End Repair Control 2* A-Tailing Control Ligation Control 5' overhang at 1 end, 3' overhang at other end Blnt with 5'-OH grop Blnt with 5'-phosphate grop Single-base 3' 'A' base overhang *End Repair Control 1 and End Repair Control 2 are separate controls inclded in the End Repair Control reagent. Inline controls can be sed for varios library insert sizes. Each is provided in ladders ranging from approximately bp in 100 bp increments. Each control molecle has a niqe DNA seqence, which indicates both its fnction and size. Inline controls are sed for trobleshooting and to identify the specific mode of failre. Using controls is optional. Yo can replace inline controls with an eqal volme of RSB. Consmables and Eqipment Some items reqired depend on the workflow performed (HS or LS) and these items are specified in separate tables. The protocol has been optimized and validated sing the items listed. Comparable performance is not garanteed when sing alternate consmables and eqipment. Consmables Consmable Spplier 1.5 ml RNase/DNase-free nonsticky tbes Thermo Fisher Scientific, part # AM µl barrier pipette tips General lab spplier 10 µl mltichannel pipettes General lab spplier 10 µl single channel pipettes General lab spplier 1000 µl barrier pipette tips General lab spplier 1000 µl mltichannel pipettes General lab spplier 1000 µl single channel pipettes General lab spplier 200 µl barrier pipette tips General lab spplier 200 µl mltichannel pipettes General lab spplier 200 µl single channel pipettes General lab spplier 96-well storage plates, rond well, 0.8 ml ('midi' plate) Thermo Fisher Scientific, part # AB-0859 Agencort AMPre XP 60 ml kit Beckman Colter Genomics, part # A63881 Agilent DNA 1000 Kit Agilent Technologies, part # Ethanol 200 proof (absolte) for moleclar biology (500 ml) Sigma-Aldrich, part # E7023 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 29

33 TrSeq Stranded mrna Reference Gide Consmable Microseal 'B' adhesive seals Nclease-free ltrapre water RNaseZap (to decontaminate srfaces) RNase/DNase-free 8-tbe strips and caps RNase/DNase-free mltichannel reagent reservoirs, disposable Spplier Bio-Rad, part # MSB-1001 General lab spplier General lab spplier General lab spplier VWR, part # SperScript II Reverse Transcriptase (1 per 48 reactions) Thermo Fisher Scientific, part # Tris-HCl 10 mm, ph8.5 Tween 20 [Optional - to aliqot reagents] 96-well 2 ml deep well plates [Optional - to determine inpt RNA integrity] Certified low range ltra agarose General lab spplier Sigma, part # P7949 Thomson Instrment Company, part # Bio-Rad, part # [Optional - positive control] Hman UHR total RNA Agilent Technologies, part # [Optional - for starting material qality assessment] One of the following: Standard Sensitivity RNA Analysis Kit (20nt Lower Marker) Agilent RNA 6000 Nano Kit Consmables for HS Workflow Advanced Analytical Technologies, part # DNF-489 Agilent Technologies, part # Consmable 96-well Hard-Shell 0.3 ml PCR plate Microseal 'A' film Spplier Bio-Rad, part # HSP-9601 Bio-Rad, part # MSA-5001 Consmables for LS Workflow Consmable 96-well 0.3 ml PCR plates Spplier General lab spplier Eqipment Eqipment 96-well thermal cycler (with programmable heated lid) One of the following: Fragment Analyzer Atomated CE System 2100 Bioanalyzer Desktop System Magnetic stand-96 Microplate centrifge Vortexer Spplier/Description General lab spplier Advanced Analytical Technologies, part # FSv2-CE2 or FSv2-CE10 Agilent Technologies, part # G2940CA Thermo Fisher Scientific, part # AM10027 General lab spplier General lab spplier Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 30

34 TrSeq Stranded mrna Reference Gide Eqipment for HS Workflow Consmable Spplier High-Speed Microplate Shaker VWR, catalog # (110 V/120 V) or (230 V) Midi plate insert for heating system Note: Two inserts are recommended to spport sccessive heating procedres. Stroboscope One of the following: Note: Two systems are recommended to spport sccessive heating procedres. Illmina, catalog # BD General lab spplier SciGene TrTemp Heating System Hybex Microsample Incbator Illmina, catalog # SC (115 V) or SC (220 V) SciGene, catalog # (115 V) or (230 V) Index Adapter Seqences For information on index adapter seqences, see Illmina Adapter Seqences (docment # ) which provides information regarding the ncleotide seqences that comprise Illmina oligoncleotides sed in Illmina seqencing technologies. Acronyms Acronym ALP ATL BBB BWB CAP CCP CDP CTA CTE CTL DCT ELB FPF FSA HS IEM LIG Definition Adapter Ligation Plate A-Tailing Mix Bead Binding Bffer Bead Washing Bffer Clean Up ALP Plate cdna Clean Up Plate cdna Plate A-Tailing Control End Repair Control Ligation Control Dilted Clster Template Eltion Bffer Fragment, Prime, Finish Mix First Strand Synthesis Act D Mix High Sample Illmina Experiment Manager Ligation Mix Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 31

35 TrSeq Stranded mrna Reference Gide Acronym LRM LS PCR PDP PMM PPC RBP RFP RPB RSB SSM STL TSP Definition Local Rn Manager Low Sample Polymerase Chain Reaction Pooled Diltion Plate PCR Master Mix PCR Primer Cocktail RNA Bead Plate RNA Fragmentation Plate RNA Prification Beads Resspension Bffer Second Strand Master Mix Stop Ligation Bffer Target Sample Plate Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 32

36 Appendix B Alternate Fragmentation Protocols Alternate Fragmentation Protocols Introdction 33 Modify RNA Fragmentation Time 33 Introdction Fragmentation of the ncleic acids is reqired for optimal library preparation, clstering, and seqencing. The TrSeq Stranded mrna Library Prep fragmentation protocol for transcriptome analysis is performed on the RNA after mrna prification sing elevated temperatres. The fragmentation reslts in libraries with inserts ranging from bp, with a median size of 150 bp. Some stdies reqire larger insert sizes, sch as splice variant analysis stdies. Modified the fragmentation times allow for varying the insert size of yor library. Modify RNA Fragmentation Time To modify the fragmentation of the RNA to allow for longer RNA fragments, the time of fragmentation can be shortened dring Prify and Fragment mrna on page 9. Modify the thermal cycler Eltion 2 Frag Prime program: 94 C for X mintes followed by a 4 C hold for the thermal cycler. Determine X based on the length of the desired RNA. See Table 3 for a range of sggested times and sizes. Table 3 Library Insert Fragmentation Time Time at 94 C (mintes) Range of Insert Length a (bp) Median Insert Length a (bp) Average Final Library Size (Bioanalyzer bp) 0 b Covaris c a. Insert length determined after clstering and seqencing with a paired-end seqencing rn. b. Skip the Incbate RFP procedre (fragmentation) for samples reqiring 0 mintes fragmentation time. Instead, place the sealed plate on the preheated thermal cycler. Close the lid and incbate the plate at 80 C for 2 mintes to elte fll length RNA from the RNA prification beads. Then, immediately place the plate on the magnetic stand and proceed to the Synthesize First Strand cdna process. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 33

37 TrSeq Stranded mrna Reference Gide Figre 14 Shortened Fragmentation Time Reslts NOTE The discrepancy between the reported insert size sing the Agilent Bioanalyzer and the insert size determined after clstering and seqencing with a paired-end seqencing rn is de to the bias towards clstering smaller fragments. To target a specific fragment size, a gel size selection step is reqired after adapter ligation. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 34