Globin Block Modules for QuantSeq Instruction Manual

Globin Block Modules for QuantSeq Instruction Manual Catalog Numbers: 070 (RS-Globin Block, Homo sapiens, 96 rxn) 071 (RS-Globin Block, Sus scrofa, 96 rxn) 015 (QuantSeq 3 mrna-seq Library Prep Kit for Illumina (FWD)) 016 (QuantSeq 3 mrna-seq Library Prep Kit for Illumina (REV) with Custom Sequencing Primer) 020 (PCR Add-on Kit for Illumina) 022 (Purification Module with Magnetic Beads) 047 (i5 Dual Indexing Add-on Kit for QuantSeq/SENSE for Illumina) 070IM154V0100

1. Overview This instruction manual outlines the protocol for using the Globin Block Modules for QuantSeq, with the QuantSeq 3 mrna-seq Library Prep Kits for Illumina (FWD, Cat. No. 015, and REV, Cat. No. 016). QuantSeq uses total RNA as input with oligo(dt) priming to generate first strand cdna. RNA removal is then performed and second strand synthesis is initiated by random priming. Final library amplification by PCR adds complete Illumina-compatible sequencing adapters and unique indices. For more detailed information about these protocols, please refer to the complete QuantSeq 3 mrna-seq User Guide available for download at our website (www.lexogen.com). Lexogen s Globin Block Modules block the generation of library fragments from the abundant and highly stable globin mrnas that are present in whole blood. In mammals, the most abundant globin mrnas are transcribed from the haemoglobin alpha and beta globin chain genes (HBA1, HBA2, and HBB). The Globin Block Modules consist of a modified RNA Removal Solution (RS-Globin Block), containing species-specific oligos complimentary to these highly abundant globin mrnas. The RS-Globin Block solutions (RS-GB ) replace the standard RNA Removal Solution (RS ) at the RNA removal step of the standard QuantSeq 3 mrna-seq Library Prep protocol. The oligos bind to the first strand cdna and prevent the generation of amplifiable library fragments from globin mrnas during second strand synthesis. Globin Block Modules are available for human (RS-Globin Block, Homo sapiens, Cat. No. 070.96) and pig (RS-Globin Block, Sus scrofa, Cat. No. 071.96) and contain enough volume for 96 reactions. These Globin Block modules can only be used with the QuantSeq 3 mrna-seq Library Prep Kits from Lexogen. 2 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

LIBRARY GENERATION 200 min 60 min Reverse Transcription 5 Total RNA 3 (AAAAAAAA) 3 (TTTTTTTTT) 5 Non-Globin mrnas Globin mrnas + RS-Globin Block Globin Blocker RNA Removal 3 (TTTTTTTTT) 3 (TTTTTTTTT) 5 5 Random Priming Random Priming Second Strand Synthesis 3 5 3 3 5 Globin Blocker 5 5 Tagged double-stranded cdna library Non-amplifiable globin cdna Purification LIBRARY AMPLIFICATION 70 min 45 min PCR Globin depleted cdna library with adapters for Illumina sequencing Purification i5 index (optional) i7 index Figure 1. Schematic overview of the QuantSeq workflow using Globin Block Modules for globin depletion. Whole blood total RNA contains a mixture of both globin and non-globin mrnas, which are both reverse transcribed by oligo(dt) priming during first strand synthesis. The RS-Globin Block (RS-GB ) solution is then added instead of the standard RNA Removal Solution (RS ). Following RNA removal, the Globin Blocker oligos bind specifically to globin first strand cdna, downstream of random primers and block globin second strand cdna synthesis. After second strand synthesis the sample will contain both non-amplifiable globin cdna fragments and tagged double-stranded cdna library fragments for non-globin mrnas. During PCR, only the non-globin library fragments are amplified to add full-length adapters and indices for Illumina sequencing. LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 3

2. Module Components and Storage Conditions Reagent Box (-20 o C) RS-GB Figure 2. Location of kit components. 2.1 RS-Globin Block, Homo sapiens, 96 rxn, Cat. No. 070.96 RS-Globin Block, Homo sapiens, 96 rxn Cat. No. 070.96 Kit Component Tube Label Volume 96 rxn Storage Removal Solution-Globin Block, Homo sapiens RS-GBHs 528 µl -20 C 2.2 RS-Globin Block, Sus scrofa, 96 rxn, Cat. No. 071.96 RS-Globin Block, sus scrofa, 96 rxn Cat. No. 071.96 Kit Component Tube Label Volume 96 rxn Storage Removal Solution-Globin Block, Sus scrofa RS-GBSs 528 µl -20 C ATTENTION: Upon receipt the solution should be stored in a -20 C freezer. NOTE: For additional user-supplied consumables and equipment needs, please refer to the QuantSeq 3 mrna-seq Library Prep Kit for Illumina User Guide. 4 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

3. Detailed Protocol 3.1 Library Generation Preparation First Strand cdna Synthesis FS1 FS2 E1 RNA Removal Second Strand Synthesis Purification thawed at RT RS-GB* thawed at RT SS1 thawed at 37 C thawed at RT SS2 thawed at RT keep on ice E2 keep on ice or at -20 C or at -20 C Thermocycler 96-well PCR plate PCR sealing films Plate centrifuge 85 C, 3 min 42 C, 15 min Thermocycler PCR sealing films Plate centrifuge 95 C, 10 min cool down to 25 C Thermocycler PCR sealing films Plate centrifuge 98 C, 1 min, then cool to 25 C (0.5 C/sec) 25 C, 30 min 25 C, 15 min PB stored at +4 C PS stored at +4 C 80 % EtOH provided by user prepare fresh! EB stored at +4 C 96-well magnetic plate 96-well PCR plate PCR sealing films * Use RS-GBHs for human (Homo sapiens, Cat. No. 070.96), and RS-GBSs for pig (Sus scrofa, Cat. No. 071.96). First Strand cdna Synthesis - Reverse Transcription An oligo(dt) primer containing an Illumina-compatible sequence at its 5 end is hybridized to the RNA and reverse transcription is performed. 1 2 3 Mix 50 ng of total RNA from whole or leukocyte blood, in a volume of 5 µl, with 5 µl of First Strand cdna Synthesis Mix 1 (FS1 ) in a PCR plate or 8-well strip tubes. If necessary, adjust the total volume to 10 µl with RNase-free water. Mix well by pipetting. Ensure the plate or PCR strips are tightly sealed and spin down to collect the liquid at the bottom of the wells. ATTENTION: For low quality RNA, prepare the total RNA in a volume of 5 µl, place the samples at room temperature, and proceed to step 3 (see also Appendix A, p.12). Denature the RNA / FS1 mix for 3 minutes at 85 C in a thermocycler and then cool down to 42 C. REMARK: Leave the reactions at 42 C until step 4. ATTENTION: Skip this step for low quality RNA. Prepare a mastermix containing 9.5 µl First Strand cdna Synthesis Mix 2 (FS2 ) and 0.5 µl Enzyme Mix 1 (E1 ) per reaction. Mix well, spin down, and pre-warm the mastermix at 42 C for 2-3 minutes. ATTENTION: For low quality RNA, prepare a mastermix containing 5 µl of First Strand cdna Synthesis Mix 1 (FS1 ), 9.5 µl FS2, and 0.5 µl E1 per sample. Mix well, spin down, and pre-warm at 42 C for 2-3 minutes. REMARK: Do not cool mastermixes! LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 5

4 Quickly spin down the denatured RNA / FS1 samples from step 2 at room temperature to make sure all liquid is collected at the bottom of the wells. Place the plate onto the thermocycler again and carefully remove the sealing foil. Add 10 µl of the FS2 / E1 mastermix to each reaction, mix by pipetting, and seal the plate. If necessary, quickly spin down the liquid at room temperature. Incubate the reactions at 42 C for 15 minutes. REMARK: For low quality RNA, add 15 µl of the pre-warmed FS1 / FS2 / E1 mastermix to each 5 µl RNA sample. Seal the plate / tubes and mix with gentle vortexing. Quickly spin down at room temperature and incubate the reactions at 42 C for 15 minutes (see Appendix A, p.12). ATTENTION: Do not cool the samples to 4 C after reverse transcription! Proceed immediately to the RNA removal step, or freeze at -20 C. RNA Removal - Globin Block During this step the RNA template is degraded, which is essential for efficient second strand synthesis, and globin blockers are added. Before removing the sealing foil after the first strand synthesis reaction, quickly spin down the plate to make sure all liquid is collected at the bottom of the wells. ATTENTION: The Removal Solution-Globin Block (RS-GB ) replaces the RNA Removal Solution (RS ) from the standard QuantSeq 3 mrna-seq Library Prep Kits for Illumina (FWD: Cat. No. 015, REV: Cat. No. 016). NOTE: RS-Globin Block, Homo sapiens (RS-GBHs ) should be used for human blood RNA libraries. RS-Globin Block, Sus scrofa (RS-GBSs ) should be used for pig blood RNA libraries. 5 6 Add 5 µl of Removal Solution-Globin Block (RS-GB ) directly to the first strand cdna synthesis reaction. Mix well and reseal the plate using a fresh foil. REMARK: Use a pipette set to 15 µl for efficient mixing. Incubate 10 minutes at 95 C, then cool down to 25 C. Spin down the plate at room temperature and carefully remove the sealing foil. 6 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

Second Strand Synthesis During this step the library is converted to double-stranded cdna. Second strand synthesis is initiated by a random primer containing an Illumina-compatible linker sequence at its 5 end. A reverse complement prevents the linker sequence from taking part in the hybridization. Globin blocker oligos hybridize specifically to globin first strand cdna and block the extension of second strand cdna. This results in non-amplifiable single-stranded globin cdna fragments. NOTE: At this point we recommend placing the purification components (PB, PS, EB) for step 12 at room temperature to give them enough time to equilibrate. ATTENTION: Second Strand Synthesis Mix 1 (SS1 ) is a viscous solution and needs to be mixed thoroughly before use. Thaw at 37 C. If a precipitate is visible, incubate at 37 C, and mix until buffer components dissolve completely. Make sure SS1 is at room temperature as this facilitates accurate pipetting. 7 8 9 10 11 Add 10 µl Second Strand Synthesis Mix 1 (SS1 ) to the reaction. Mix well by pipetting, and seal the plate. REMARK: Use a pipette set to 30 µl for efficient mixing. Incubate the plate for 1 minute at 98 C in a thermocycler, and slowly cool down to 25 C by setting the ramp speed to 10 % (0.5 C/second). Incubate the reaction for 30 minutes at 25 C. Quickly spin down the plate at room temperature before removing the sealing foil. Prepare a mastermix containing 4 µl Second Strand Synthesis Mix 2 (SS2 ) and 1 µl Enzyme Mix 2 (E2 ). Mix well. Add 5 µl of the SS2 / E2 mastermix per reaction. Mix well. REMARK: Use a pipette set to 30 µl for efficient mixing. Incubate the reaction at 25 C for 15 minutes. stored at -20 C at this point. Safe stopping point. Libraries can be LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 7

Purification The double-stranded library is purified by using magnetic beads to remove all reaction components. The purification components (PB, PS, EB) should equilibrate for 30 minutes at room temperature before use. Purification Beads (PB) may have settled and must be properly resuspended before adding them to the reaction. 12 13 14 15 16 17 18 19 20 21 22 23 24 Add 16 µl of properly resuspended Purification Beads (PB) to each reaction, mix well, and incubate for 5 minutes at room temperature. Place the plate onto a magnetic plate, and let the beads collect for 2-5 minutes or until the supernatant is completely clear (depends on the strength of your magnet). Remove and discard the clear supernatant without removing the PCR plate from the magnetic plate. Make sure that accumulated beads are not disturbed. Add 40 µl of Elution Buffer (EB), remove the plate from the magnet, and resuspend the beads properly in EB. Incubate for 2 minutes at room temperature. Add 56 µl of Purification Solution (PS) to the beads / EB mix to reprecipitate the library. Mix thoroughly, and incubate for 5 minutes at room temperature. ATTENTION: For low quality RNA add only 48 µl PS. Place the plate onto a magnetic plate, and let the beads collect for 2-5 minutes or until the supernatant is completely clear. Remove and discard the clear supernatant without removing the PCR plate from the magnetic plate. Make sure that accumulated beads are not disturbed. Add 120 µl of 80 % EtOH, and incubate the beads for 30 seconds. Leave the plate in contact with the magnet as beads should not be resuspended during this washing step. Remove and discard the supernatant. Repeat this washing step once for a total of two washes. Make sure to remove the supernatant completely as traces of ethanol can inhibit subsequent PCR reactions. Leave the plate in contact with the magnet, and let the beads dry for 5-10 minutes or until all ethanol has evaporated. ATTENTION: Dry the beads only at room temperature and do not let the beads dry too long (visible cracks appear), as this will negatively influence the elution and the resulting library yield. Add 20 µl of Elution Buffer (EB) per well, remove the plate from the magnet and resuspend the beads properly in EB. Incubate for 2 minutes at room temperature. Place the plate onto a magnetic plate and let the beads collect for 2-5 minutes or until the supernatant is completely clear. Transfer 17 µl of the clear supernatant into a fresh PCR plate. Make sure not to transfer any beads. Safe stopping point. Libraries can be stored at -20 C at this point. 8 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

3.2 Library Amplification - Single Indexing (i7 only) This section describes PCR for multiplexing and unique indexing of up to 96 libraries using the i7 indices included in the kit (i7 Index Plate). Lexogen also offers an i5 Dual Indexing Add-on Kit (Cat. No. 047) for unique indexing of up to 384 libraries. For details, please refer to the i5 Dual Indexing Add-on Kit Instruction Manual. Preparation PCR PCR thawed at RT E3 keep on ice or at -20 C i7 Index Plate thawed at RT; spin down before opening! 96-well PCR plate PCR sealing films Plate centrifuge Thermocycler 98 C, 30 sec 98 C, 10 sec 65 C, 20 sec 72 C, 30 sec 72 C, 1 min 10 C, 13-17x (See Appendix A, p.12) Purification PB stored at +4 C PS stored at +4 C 80 % EtOH provided by user; prepare fresh! EB stored at +4 C 96-well magnetic plate 96-well PCR plate Plate centrifuge PCR sealing films Endpoint PCR The library is amplified to add the complete adapter sequences required for cluster generation and unique i7 indices, and to generate sufficient material for quality control and sequencing. ATTENTION: Libraries made from blood total RNA using RS-Globin Block for globin depletion may require an additional PCR cycle compared to libraries prepared with the standard RNA Removal Solution (for guidelines, see Appendix A, p.12). However, as blood RNA samples may vary in terms of origin and quality, we strongly recommend performing a qpcr to determine the optimal number of cycles for the library amplification (see Appendix B, p.13). NOTE: At this point we recommend placing the purification components (PB, PS, EB) for step 29 at room temperature to give them enough time to equilibrate. 25 Prepare a mastermix containing 7 µl of PCR Mix (PCR ) and 1 µl Enzyme Mix 3 (E3 ) per reaction. 26 Add 8 µl of this PCR / E3 mastermix to 17 µl of the eluted library. LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 9

27 28 Add 5 µl of the respective i7 index (7001-7096, in 96-well plate). Mix well by pipetting. Seal the PCR plate and quickly spin down to make sure all liquid is collected at the bottom of the well. ATTENTION: Spin down the i7 Index Plate before opening! Visually check fill levels. Pierce or cut open the sealing foil of the wells containing the desired indices. Avoid cross contamination! Reseal opened wells of the i7 primer plate after usage to prevent cross contamination! Conduct 13-17 cycles of PCR (see Appendix A, p.12) with: Initial denaturation at 98 C for 30 seconds, 13-17 cycles of 98 C for 10 seconds, 65 C for 20 seconds and 72 C for 30 seconds, and a final extension at 72 C for 1 minute, hold at 10 C. Safe stopping point. Libraries can be stored at -20 C at this point. Purification The finished library is purified from PCR components that can interfere with quantification. The Purification Beads (PB) may have settled and must be properly resuspended before adding them to the reaction. 29 30 31 32 33 34 35 36 37 Add 30 µl of properly resuspended Purification Beads (PB) to each reaction, mix well, and incubate for 5 minutes at room temperature. ATTENTION: For low quality RNA add only 27 µl PB. Place the plate onto a magnetic plate, and let the beads collect for 2-5 minutes or until the supernatant is completely clear. Remove and discard the clear supernatant without removing the PCR plate from the magnetic plate. Make sure that accumulated beads are not disturbed. Add 30 µl of Elution Buffer (EB), remove the plate from the magnet, and resuspend the beads properly in EB. Incubate for 2 minutes at room temperature. Add 30 µl of Purification Solution (PS) to the beads / EB mix to reprecipitate the library. Mix thoroughly, and incubate for 5 minutes at room temperature. Place the plate onto a magnetic plate, and let the beads collect for 2-5 minutes or until the supernatant is completely clear. Remove and discard the clear supernatant without removing the PCR plate from the magnetic plate. Make sure that accumulated beads are not disturbed. Add 120 µl of 80 % EtOH, and incubate the beads for 30 seconds. Leave the plate in contact with the magnet as beads should not be resuspended during this washing step. Remove and discard the supernatant. Repeat this washing step once for a total of two washes. Make sure to remove the supernatant completely. 10 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

38 39 40 41 Leave the plate in contact with the magnet, and let the beads dry for 5-10 minutes or until all ethanol has evaporated. ATTENTION: Dry the beads only at room temperature and do not let the beads dry too long (visible cracks appear), as this will negatively influence the elution and the resulting library yield. Add 20 µl of Elution Buffer (EB) per well, remove the plate from the magnet, and resuspend the beads properly in EB. Incubate for 2 minutes at room temperature. Place the plate onto a magnetic plate and let the beads collect for 2-5 minutes or until the supernatant is completely clear. Transfer 15-17 µl of the supernatant into a fresh PCR plate. Do not transfer any beads. Libraries are now finished and ready for quality control, pooling, and cluster generation. NOTE: For further information on quality control, multiplexing, sequencing, and data analysis, please see the QuantSeq 3 mrna-seq Library Prep Kit for Illumina User Guide. Safe stopping point. Libraries can be stored at -20 C at this point. LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 11

4. Appendix A: Input RNA and PCR Cycles Total RNA from blood is the intended input for QuantSeq library prep using RS-Globin Block solutions for globin depletion. No prior depletion of globin mrna, poly(a) enrichment, or ribosomal RNA is required. The minimum recommended input for this protocol is 50 ng of total RNA from whole blood, or leukocyte-enriched blood samples (RIN 6). It is important to ensure that total RNA is of high purity: Ideal values for A260/280 and A260/230 absorbance ratios should be 1.8-2.1, and approximately 2, respectively. For low quality RNA samples, please follow the protocol modification listed at the bottom of the page. We recommend the use of Lexogen s SPLIT RNA Extraction Kit (Cat. No. 008) for isolation of total RNA from fresh or frozen blood. Additional red blood cell lysis may be performed prior to RNA extraction to obtain leukocyte-enriched blood RNA. For protocol details please see: www. lexogen.com/split-rna-extraction. Total RNA isolated from blood using the PAXgene Blood RNA System (Tubes and Kit, Qiagen*) is also compatible with this protocol. The table below shows an example of PCR cyle numbers used for input amounts of human and pig total RNA from blood. Species Whole Blood or Leukocyte Blood Input Amount Undepleted PCR Cycles + RS-Globin Block Human Whole blood Leukocyte-enriched blood 250 ng 13 14 50 ng 15 16 50 ng 16 17 50 ng* 15 16 Pig Whole blood 100 ng 16 17 * Indicates RNA isolated using PAXgene Blood System (Tubes and Kit, Qiagen), which includes 24-hour red blood cell lysis. All other RNA was extracted using the SPLIT RNA Extraction Kit (Cat. No. 008.48). All libraries were prepared with single indexing. Linker sequences are 122 bp including 6 nt long i7 indices. Globin mrnas constitute 50-80 % of the total mrna pool. Therefore, when blocking these mrnas from the final step of library generation, the cycle numbers need to be increased to achieve the same library yield as non-depleted samples. Adding one additional PCR cycle compared to non-depleted blood samples is typically sufficient. However, as the mrna content, purity, and quality of total RNA from blood samples may vary depending on the origin of the sample. Therefore, we strongly recommend performing the qpcr assay to determine the optimal number of cycles for the library amplification (see Appendix B, p.13). ATTENTION: For low quality RNA we recommend: skipping step 2, reducing the amount of PS added in step 16 (48 µl instead of 56 µl), and reducing the amount of PB in step 29 (27 µl instead of 30 µl). For information regarding the use of input amounts <50 ng please contact info@lexogen.com, or see the QuantSeq FWD online FAQs at www.lexogen.com. * Note: PAXgene is a registered trademark of Qiagen Group. 12 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

5. Appendix B: qpcr and Reamplification The mrna content and quality of total RNA affects the number of PCR cycles needed for the final library amplification step. We strongly recommend taking advantage of the qpcr assay to determine optimal cycle numbers for library amplification, particularly when preparing libraries with RS-Globin Block. The depletion of globin mrnas removes a major proportion of the library and this can affect the number of PCR cycles required. Over- or undercycling may bias your sequencing results (transcript abundance estimation and library quantification) and can be avoided by optimizing the PCR cycle number. The PCR Add-on Kit for Illumina (Cat. No. 020.96) provides additional PCR reagents and the P7 Primer (7000 ) needed for the qpcr assay. In addition, SYBR Green I nucleic acid stain (10,000x in DMSO, Sigma Aldrich, Cat. No. S9430) is required (user-provided). The use of SYBR Green qpcr mastermixes is not recommended. qpcr to Determine the Exact Cycle Number of Your Endpoint PCRs To determine the cycle number of your endpoint PCR, dilute the double-stranded library from step 24 to 19 µl by adding 2 µl Elution Buffer (EB). Add 1.7 µl of the cdna into a PCR containing 7 µl PCR Mix (PCR ), 5 µl P7 Primer (7000 ), and 1 µl Enzyme Mix (E ) from the PCR Add-on Kit (Cat. No. 020.96). Store the remaining cdna at -20 C. To enable quantification, SYBR Green I (or an equivalent fluorophore) must be added to a final concentration of 0.1x. For 0.1x SYBR Green I, add 1.2 µl of a 2.5x SYBR Green I solution (1:4,000 SYBR Green I dilution, diluted in DMSO). Fill up the total PCR reaction volume to 30 µl with EB. Alternatively, if 8 qpcrs are run at the same time, best practice would be to prepare a mastermix with 0.15 µl of a 20x SYBR Green I solution (1:100 SYBR Green I dilution, in DMSO) per reaction. Include a no template control! SYBR Green I has an emission maximum at 520 nm, which for some qpcr machines have to be adjusted manually. Perform 40-50 cycles of PCR (see p.9 for PCR Program) using a real-time PCR machine. Determine the value at which the fluorescence reaches the plateau. Calculate where the fluorescence is at 50 % of the maximum and determine at which cycle this 50 % of fluorescence value is reached. As the endpoint PCR will contain 10x more cdna compared to the qpcr, subtract three from this cycle number. This is then the final cycle number you should use for the endpoint PCR with the remaining 17 µl of the template. There is no need to purify or analyze the overcycled PCR reaction on a Bioanalyzer. EXAMPLE: 50 ng total RNA input was used for generating libraries. Using 1.7 µl of cdna for a qpcr, the cycle number corresponding to 50 % of the maximum fluorescence was 19 cycles. The remaining 17 µl of the template should therefore be amplified with 16 cycles (19-3 cycles = 16 cycles). REMARK: The qpcr assay can be used for dual or single indexed libraries. Reamplification of i7 Indexed Libraries (i7 only) Lexogen s PCR Add-on Kit (Cat. No. 020.96) also contains a Reamplification Primer (RE ) that can be used to reamplify single indexed (i7) libraries to get enough material for sequencing if they LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 13

were undercycled. For details please refer to the PCR Add-on Kit Instruction Manual. ATTENTION: Do not use 7000 for the reamplification of i7 indexed libraries! This will lead to a loss of indices and to a mixed and not assignable sequence pool in the NGS run. ATTENTION: Do not use the Reamplification Primer (RE ) for a qpcr assay on the cdna-library as the cdna lacks binding sites for the Reamplification Primer. RE can be only used on i7 indexed, amplified PCR libraries. For reamplification of dual indexed libraries contact Lexogen at info@lexogen.com. 14 LEXOGEN Globin Block Modules for QuantSeq Instruction Manual

6. Appendix C: Revision History Publication No. / Revision Date 070IM154V0100 Dec. 19, 2017 Change Initial Release: Homo sapiens and Sus scrofa Globin Block Modules. Page LEXOGEN Globin Block Modules for QuantSeq Instruction Manual 15

Globin Block Modules for QuantSeq Instruction Manual Lexogen GmbH Campus Vienna Biocenter 5 1030 Vienna, Austria Telephone: +43 (0) 1 345 1212-41 Fax: +43 (0) 1 345 1212-99 E-mail: info@lexogen.com Lexogen GmbH, 2018