USER GUIDE. Universal Plus mrna-seq PART NO. 0508, 9133, 9134

Transcription

1 USER GUIDE PART NO. 0508, 9133, 9134

2 Patents, Licensing and Trademarks 2017 NuGEN Technologies, Inc. All rights reserved. The Encore, Ovation and Applause families of products and methods of their use are covered by several issued U.S. and International patents and pending applications ( NuGEN, Ovation, SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners. The purchase of this product conveys to the buyer the limited, non-exclusive, non-transferable right (without the right to modify, reverse engineer, resell, repackage or further sublicense) under these patent applications and any patents issuing from these patent applications to use this product and methods, accompanying this user guide, for research and development purposes solely in accordance with the intended use described and the written instructions provided in this user guide. No license to make or sell products by use of this product is granted to the buyer whether expressly, by implication, by estoppels or otherwise. In particular, the purchase of this product does not include or carry any right or license to use, develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics. For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research, please contact NuGEN Technologies, Inc., 201 Industrial Road, Suite 310, San Carlos, CA Phone or ; FAX or Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase, provided that the product is handled and stored according to published instructions, and that the product is not altered or misused. If the product fails to meet these performance standards, NuGEN will replace the product free of charge or issue a credit for the purchase price. NuGEN s liability under this warranty shall not exceed the purchase price of the product. NuGEN shall assume no liability for direct, indirect, consequential or incidental damages arising from the use, results of use or inability to use its products. NuGEN reserves the right to change, alter or modify any product to enhance its performance and design. NuGEN s products are developed, designed and sold FOR RESEARCH USE ONLY. This product is not to be used for diagnostic or therapeutic purposes, nor is it to be administered to humans or animals. Except as expressly set forth herein, no right to modify, reverse engineer, distribute, offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NuGEN product. The buyer agrees to use NuGEN products accompanying the product insert in accordance with the intended use and the written instructions provided.

3 Table of Contents Contents I. Introduction... 1 A. Background... 1 B. Workflow... 1 C. Performance Specifications... 2 D. Quality Control... 2 E. Storage and Stability... 3 F. Safety Data Sheet (SDS)... 3 G. Before You Start... 3 II. Kit Components... 4 A. Reagents Provided... 4 B. Additional Equipment, Reagents and Labware... 6 III. Planning the Experiment... 9 A. Input RNA Requirements... 9 B. Working with the 32- and 96-Plex Adaptor Plates... 9 C. Amplified Library Storage D. Sequencing Recommendations and Guidelines E. Data Analysis IV. Overview A. Overview B. Protocol Notes C. Agencourt Beads D. Programming the Thermal Cycler V. Protocol A. Poly(A) Selection B. RNA Fragmentation C. First Strand cdna Synthesis D. Second Strand cdna Synthesis E. cdna Purification F. End Repair G. Adaptor Ligation H. Strand Selection I. Strand Selection Purification J. Library Amplification A K. Probe Binding (optional AnyDeplete workflow) L. Targeted Depletion (optional AnyDeplete workflow) M. Library Amplification B (optional AnyDeplete workflow) N. Amplified Library Purification O. Quantitative and Qualitative Assessment of the Library... 30

4 Table of Contents VI. Technical Support VII. Appendix A. Barcode Sequences and Guidelines for Multiplex Experiments B. Frequently Asked Questions (FAQs)... 33

5 I. Introduction A. Background is an end-to-end solution for generation of RNA-Seq libraries derived from poly(a) selected RNA. This kit is compatible with inputs from 10 ng 1 µg of high quality total RNA obtained from a broad range of tissues or cell lines. Using paramagnetic beads coupled with oligo d(t), poly(a) transcripts including mrna are isolated from total RNA prior to cdna synthesis and DimerFree library construction. This kit also features an optional integration of AnyDeplete (formerly InDA-C) for depletion of unwanted transcript sequences, providing more efficient use of sequencing resources. The reagents for AnyDeplete are provided only with purchase of 9133 (depletion of human globin sequences) and 9134 (depletion of human mitochondrial sequences). B. Workflow As shown in Figure 1, the workflow consists of poly(a) RNA selection, RNA fragmentation and double-stranded cdna generation using a mixture of random and oligo(dt) priming, followed by end repair to generate blunt ends, adaptor ligation, strand selection, and PCR amplification to produce the final library. An optional AnyDeplete workflow may be performed prior to library amplification for removal of unwanted transcripts. The entire workflow can be completed in one day, and yields cdna libraries ready for either single read or paired-end sequencing on Illumina sequencing platforms. Page 1 of 37 M01442 v2

6 I. Introduction Figure 1. Workflow. Total RNA Poly(A) RNA Selection RNA Fragmentation First Strand Synthesis Second Strand Synthesis End Repair Adaptor Ligation Strand Selection OPTIONAL AnyDeplete Library Amplification Sequencing-ready cdna Libraries C. Performance Specifications is designed to generate mrna-seq libraries suitable for either single-read or paired-end sequencing on Illumina NGS platforms, starting with as little as 10 ng total RNA. D. Quality Control Every lot of undergoes functional testing to meet specifications for library generation performance. Page 2 of 37 M01442 v2

7 I. Introduction E. Storage and Stability is provided as two shipments. The core library preparation kit and optinal AnyDeplete module are shipped on dry ice and the poly(a) selection reagents are shipped at 4 C. Both packages should be unpacked immediately upon receipt. Note: This product contains components with multiple storage temperatures. Oligo d(t) Beads, mrna Elution Buffer, mrna Wash Buffer, and mrna Binding Buffer should be stored at 4 C. K562 Control RNA (RNA ver 1) should be stored at 80 C. All remaining components should be stored at 20 C in a freezer without a defrost cycle. This product has been tested to perform to specifications after as many as six freeze/ thaw cycles. Kits handled and stored according to the above guidelines will perform to specifications for at least six months. F. Safety Data Sheet (SDS) If appropriate, an SDS for this product is available on the NuGEN website at G. Before You Start Please review this User Guide before using this kit for the first time, including the Kit Components, Planning the Experiment, Overview, Protocol and FAQ sections. For more information, visit the page at NuGEN.com ( New to NGS? Contact NuGEN Technical Support at techserv@nugen.com for tips and tricks for getting started. Page 3 of 37 M01442 v2

8 II. Kit Components A. Reagents Provided The (Part No. 0508) is a bundle of Part No and The AnyDeplete module (Part No. 0359) and AnyDeplete probe mix (listed in Tables Table 3 and Table 4) are provided only with purchase of Part No (depletion of human globin sequences) and Part No (depletion of human mitochondrial sequences). Table 1. (Part No. 0358) COMPONENT PART NUMBER PART NUMBER PART NUMBER VIAL LABEL VIAL NUMBER 2x Fragmentation Buffer S02352 S02351 S02388 Clear FB1 ver 1 Actinomycin D S02350 S02349 S02379 Brown First Strand Buffer Mix First Strand Enzyme Mix Second Strand Buffer Mix Second Strand Enzyme Mix End Repair Buffer Mix End Repair Enzyme Mix End Repair Enhancer S02329 S02330 S02377 Blue A2 ver 14 S02331 S02201 S02378 Blue A3 ver 6 S02332 S02333 S02380 Yellow B1 ver 11 S01508 S01531 S02055 Yellow B2 ver 4 S02334 S01708 S02381 Blue ER1 ver 7 S01510 S01533 S02382 Blue ER2 ver 4 S01862 S01709 S02383 Blue ER3 ver 2 Ligation Buffer Mix S01936 S02338 S01689 Yellow L1 ver 4 Ligation Enzyme Mix Strand Selection Buffer Mix I Strand Selection Enzyme Mix I S01467 S01535 S02384 Yellow L3 ver 4 S01511 S01710 S02393 Purple SS1 S01512 S01537 S02385 Purple SS2 Page 4 of 37 M01442 v2

9 II. Kit Components Table 1 (Part No. 0358), continued COMPONENT PART NUMBER PART NUMBER PART NUMBER VIAL LABEL VIAL NUMBER Strand Selection Enzyme Mix II Amplification Buffer Mix Amplification Primer Mix Amplification Enzyme Mix S01514 S01539 S02386 Purple SS4 S01871 S01642 S01893 Red P1 ver 4 S02179 S01767 S02063 Red P2 ver 10 S02180 S01644 S02387 Red P3 ver 2 K562 Control RNA S02289 S02289 S02289 Clear RNA ver 1 32-Plex Ligation Adaptor Plate 96-Plex Ligation Adaptor Plate Nuclease-free Water S02258 L2V22DR-BC S02259 L2V22DR-BC S01001 S01113 S01113 Green D1 Ligation Adaptor Mix S02341 S02342 S02343 S02344 S02345 S02346 S02347 S02348 Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow L2V22DR-BC1 L2V22DR-BC2 L2V22DR-BC3 L2V22DR-BC4 L2V22DR-BC5 L2V22DR-BC6 L2V22DR-BC7 L2V22DR-BC8 Table 2. Ovation mrna-seq Module (Part No. 0408) COMPONENT PART NUMBER PART NUMBER PART NUMBER VIAL LABEL VIAL NUMBER Oligo dt Beads S02357 S02244 S02248 Clear mrna Elution Buffer S02358 S02243 S02247 Clear mrna Wash Buffer S02359 S02242 S02246 Clear Page 5 of 37 M01442 v2

10 II. Kit Components mrna Binding Buffer S02360 S02241 S02245 Clear Table 3. AnyDeplete Module (Part No. 0359) COMPONENT PART NUMBER PART NUMBER PART NUMBER VIAL LABEL VIAL NUMBER AnyDeplete Buffer Mix S02291 S02306 S02390 Purple AD1 ver 1 AnyDeplete Probe Mix AnyDeplete Enzyme Mix I AnyDeplete Enzyme Mix II See Table 4 for available probe mixes S02292 S02307 S02391 Purple AD2 ver 1 S02293 S02308 S02392 Purple AD3 ver 1 Table 4. AnyDeplete Probe Mix COMPONENT PART NUMBER VIAL LABEL VIAL NUMBER Universal Plus AnyDeplete Probe Mix Human Globin Universal Plus AnyDeplete Probe Mix Human Mito S02364 Purple AD14 ver 1 S02367 Purple AD15 ver 1 Note: The reagents in are similar to reagents in our other kits; however, unless the component part numbers are identical, these reagents do not have exactly the same composition and, therefore, are not interchangeable. Do not exchange or replace one reagent named, for example, A1 with another A1, as it will adversely affect performance. Page 6 of 37 M01442 v2

11 II. Kit Components B. Additional Equipment, Reagents and Labware Required Materials Equipment Agilent 2100 Bioanalyzer or 2200 TapeStation Instrument, or other equipment for electrophoretic analysis of nucleic acids Microcentrifuge for individual 1.5 ml and 0.5 ml tubes Microcentrifuge for 0.2 ml tube strips or plates µl pipette, 2 20 µl pipette, µl pipette, µl pipette 2 20 µl or 5 50 µl multichannel pipette and µl or µl multichannel pipette for sample mixing Vortexer Thermal cycler with 0.2 ml tube heat block, heated lid, and 100 µl reaction capacity Qubit 2.0 or 3.0 Fluorometer (Thermo Fisher Scientific) or appropriate fluorometer and accessories for quantification of fragmented DNA and amplified libraries. Reagents Agencourt RNAClean XP Beads or AMPure XP Beads (Beckman Coulter, Cat. #A63987 or A63881) Low-EDTA TE Buffer, 1X, ph 8.0 (Alfa Aesar, Cat. #J75793) optional; for diluting nucleic acids Ethanol (Sigma-Aldrich, Cat. #E7023), for purification steps Nuclease-free water (Affymetrix, Cat. #71786), for purification steps Agilent High Sensitivity DNA Kit (Agilent, Cat. # ) or equivalent EvaGreen, 20X (Biotium, Cat. #31000) optional; for optimizing Library Amplification with qpcr Supplies and Labware Nuclease-free pipette tips 1.5 ml and 0.5 ml RNase-free microcentrifuge tubes 0.2 ml PCR strip tubes or 0.2 ml thin-wall PCR plates Low-retention microcentrifuge tubes (DNA LoBind Tubes, Eppendorf Cat# or ) Magnetic stand for 0.2 ml strip tubes or plates. (Beckman Coulter Cat. #A29164 or A32782; Thermo Fisher Scientific Cat. #12331D, 12027, or 12332D; Promega Cat. #V8351; others). Other magnetic stands may be used as well, although their performance has not been validated by NuGEN. Cleaning solutions such as RNaseZap RNase Decontamination Solution (Thermo Fisher Scientific, Cat. #AM9780) and DNA OFF (MP Biomedicals, Cat. #11QD0500) 96-well plate sealing foil (Thermo Fisher Scientific, Cat. #AB1720) Disposable gloves Kimwipes Ice bucket Page 7 of 37 M01442 v2

12 II. Kit Components To Order Alfa Aesar, Agilent, Beckman Coulter, Biotium, Eppendorf, MP Biomedicals, Promega, Sigma-Aldrich, Inc., Thermo Fisher Scientific, Page 8 of 37 M01442 v2

13 III. Planning the Experiment A. Input RNA Requirements 1. RNA Quantity Total RNA input must be between 10 ng 1 µg. Inputs outside of these ranges may affect reaction stoichiometry, resulting in sub-optimal libraries. Lower input amounts will potentially result in insufficient yields depending on the requirements of the analytical platform. We strongly recommend quantification of total RNA to ensure the minimum input requirement is met. 2. RNA Purity RNA samples must be free of contaminating proteins and other cellular material, organic solvents (including phenol and ethanol) and salts used in many RNA isolation methods. When preparing small amounts of RNA, we recommend using a commercially available system that does not require organic solvents. If using an RNA isolation method based on organic solvents, such as TRIzol, we recommend column purification after isolation. One measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm. The A260:A280 ratio for RNA samples should be in excess of RNA Integrity The kit is designed for use with RNA samples of high molecular weight with little or no evidence of degradation. RNA integrity can be determined using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano LabChip or RNA 6000 Pico LabChip. B. Working with the 32- and 96-Plex Adaptor Plates The Adaptor Plate included with the 32 and 96 reaction kit contains adaptor mixes, each with a unique eight-base barcode. Each well (A01 H04 for the 32-plex adaptor plate and A01-H12 for the 96-plex adaptor plate) contains sufficient volume for preparation of a single library. The Adaptor Plates are sealed with a foil seal designed to provide airtight storage. Thaw adaptor plate on ice and spin down. Do NOT warm above room temperature. Make sure all adaptor mixes are collected at the bottom of the wells and return the adaptor plate to ice after centrifuging. When pipetting the adaptor mixes, puncture the seal for each well you wish to use with a fresh pipet tip, and transfer the appropriate volume of adaptor into your sample. The remaining wells of the plate should remain sealed for use at a later date. Cover used wells with a new foil seal to prevent any remaining adaptor-containing liquid from contaminating future reactions. For details regarding barcode color balancing for multiplex sequencing, please see Appendix A on page 32. Page 9 of 37 M01442 v2

14 III. Planning the Experiment C. Amplified Library Storage Amplified libraries may be stored at 20 C. D. Sequencing Recommendations and Guidelines produces RNA-Seq libraries compatible with Illumina NGS platforms. These libraries should be sequenced using the Illumina protocol for multiplex sequencing, following the recommendations for the specific sequencer. Universal Plus mrna-seq libraries contain an 8 bp barcode. These barcodes differ from the sequences used by Illumina and can be found in Appendix A. Figure 2. Library Structure. Flow Cell Sequence Illumina FWD Read Primer Library Insert Illumina REV Read Primer Flow Cell Surface Illumina Index Read Primer Barcode Flow Cell Sequence E. Data Analysis Once the data have been parsed according to sample, additional sample-specific data analysis may be employed according to the requirements of the experiment. NOTE: The forward read from libraries represents the sense strand. This may be opposite to stranded data from other library preparation kits and may require slight modification of the data analysis workflow. Contact NuGEN Technical Support for more information. Page 10 of 37 M01442 v2

15 IV. Overview A. Overview is performed in the following stages: Standard workflow Optional AnyDeplete workflow 1. Poly(A) selection 60 min 60 min 2. RNA fragmentation 15 min 15 min 3. cdna synthesis a. First strand synthesis 40 min 40 min b. Second strand synthesis and purification 100 min 100 min 4. Library construction a. End repair 45 min 45 min b. Adaptor ligation 35 min 35 min c. Strand selection and purification 45 min 45 min 5. Library amplification and purification 90 min 6. AnyDeplete (optional) a. Probe binding 25 min b. Targeted depletion 40 min c. Library amplification and purification 90 min Total time to prepare amplified library 7 hr 10 min 8 hr 15 min B. Protocol Notes Controls We recommend the routine use of the provided K562 positive control RNA, especially the first time a reaction is set up. The use of a positive control RNA will establish a baseline of performance and provide the opportunity to become familiar with the bead purification step. This step may be unfamiliar to many users and can be especially prone to handling variability in using the magnet plate, so a practice run with the plate is highly recommended. Routine use of a no template control (NTC) is recommended to monitor the work environment for potential carryover contamination of previous libraries. Page 11 of 37 M01442 v2

16 IV. Overview General Workflow Set up no fewer than 4 reactions at a time (8-reaction kit) or 8 reactions at a time (32-reaction kit). This ensures sufficient reagent recoveries for the full nominal number of amplifications from the kit. Making master mixes for fewer than 4 samples at a time may affect reagent recovery volumes. Thaw components used in each step and immediately place them on ice. Always keep thawed reagents and reaction tubes on ice unless otherwise instructed. After thawing and mixing buffer mixes, if any precipitate is observed, re-dissolve the precipitate completely prior to use. You may gently warm the buffer mix for 2 minutes at room temperature followed by brief vortexing. Keep enzyme mixes on ice after briefly spinning to collect the contents. Do not vortex enzyme mixes nor warm any enzyme or primer mixes. When preparing master mixes, use the minimal amount of extra material to ensure 8 or 32 reactions in the kit. When placing small amounts of reagents into the reaction mix, pipet up and down several times to ensure complete transfer. When instructed to mix via pipetting, gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix. Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block. Reagents Use the water provided with the kit (green: D1) or an alternate source of nuclease-free water. We do not recommend the use of DEPC-treated water with this protocol. Components and reagents from other NuGEN products should not be used with this product. Use only fresh ethanol stocks to make ethanol for washes in the purification protocols. Make the ethanol mixes fresh, carefully measuring both the ethanol and water with pipettes. Lower concentrations of ethanol in wash solutions will result in loss of yield as the higher aqueous content will dissolve the cdna and wash it off the beads or column. C. Agencourt Beads Agencourt RNAClean XP or Ampure XP Beads (Agencourt beads) are suitable for use with. There are modifications to the Agencourt beads standard procedure; therefore, you must follow the protocols outlined in this user guide for the use of these beads. The bead purification process used for cdna purification before amplification consists of: 1. Binding of DNA to Agencourt beads 2. Magnetic separation of beads from supernatant 3. Ethanol wash of bound beads to remove contaminants 4. Elution Page 12 of 37 M01442 v2

17 IV. Overview Figure 3. Agencourt bead purification process. 1. Binding 2. Separation 3. Ethanol Wash 4. Elution Magnet Magnet Magnet Reproduced from original picture from Agencourt/Beckman Coulter Genomics Tips and Notes Remove Agencourt beads from 4 C and leave at room temperature for at least 30 minutes. Ensure that they have completely reached room temperature before use. Cold beads and buffer will result in reduced recovery. Fully resuspend beads by inverting and tapping before adding to the sample. Note that we recommend specific sample to bead volume ratios in our Agencourt bead protocols. In many cases our recommendations differ from the standard Beckman Coulter protocol. It is critical to let the beads separate on the magnet for the full time indicated at each step. Removing the binding buffer before the beads have completely separated will impact yields. Take care to minimize bead loss throughout the procedure. Any significant loss of beads after the binding steps and ethanol washes will impact DNA yields. After the binding step has been completed, it is important to minimize bead loss when removing the binding buffer. With the samples placed on the magnetic plate, carefully remove the specified quantity of binding buffer from each sample to avoid disturbing the beads. Ensure that the 70% ethanol wash is freshly prepared from fresh ethanol stocks. Lower percent ethanol mixes will reduce recovery. During the ethanol washes, keep the samples on the magnet. The beads should not be allowed to disperse; the magnet will keep the beads on the walls of the sample wells or tubes. It is critical that all residual ethanol be removed prior to elution. Therefore, when removing the final ethanol wash, first remove most of the ethanol, then allow the excess to collect at the bottom of the tube before removing the remaining ethanol. This reduces the required bead air-drying time. After drying the beads for 10 minutes, inspect each tube carefully and make certain that all the ethanol has evaporated before proceeding with the amplification step. It is strongly recommended that strip tubes or partial plates are firmly placed when used with the magnetic plate. We don t advise the use of individual tubes, as they are not very stable on the magnetic plates. Page 13 of 37 M01442 v2

18 IV. Overview D. Programming the Thermal Cycler Use a thermal cycler with a heat block designed for 0.2 ml tubes, equipped with a heated lid, and with a capacity of 100 μl reaction volume. Prepare the programs shown in Table 5 following the operating instructions provided by the manufacturer. For thermal cyclers with an adjustable heated lid, set the lid temperature at 100 C. For thermal cyclers with a fixed-temperature heated lid use the default settings (typically C). Table 5. Thermal Cycler Programming mrna SELECTION Program 1 Poly(A) RNA Binding Program 2 RNA Elution RNA FRAGMENTATION Program 3 RNA Fragmentation cdna SYNTHESIS VOLUME 65 C 5 min, hold at 4 C 110 μl 80 C 2 min, hold at 25 C 50 μl VOLUME 94 C 8 min, hold at 4 C 20 μl VOLUME Program 4 First Strand Synthesis 25 C 5 min, 42 C 15 min, 70 C 15 min, hold at 4 C 25 μl Program 5 Second Strand Synthesis END REPAIR Program 6 End Repair LIGATION Program 7 Adaptor Ligation STRAND SELECTION Program 8 Strand Selection 16 C 60 min, hold at 4 C 75 μl VOLUME 25 C 30 min, 70 C 10 min, hold at 4 C 15 μl VOLUME 25 C 30 min, hold at 4 C 30 μl VOLUME 72 C 10 min, hold at 4 C 100 μl Page 14 of 37 M01442 v2

19 IV. Overview Table 5 Thermal Cycler Programming, continued AMPLIFICATION (FOR STANDARD WORKFLOWS) VOLUME Program 9 Library Amplification A ANYDEPLETE (OPTIONAL) Program 10 Probe Binding 37 C 10 min, 95 C 2 min, 2x(95 C 30 s, 60 C 90 s), 15x* (95 C 30 s, 65 C 90 s), 65 C 5 min, hold at 4 C 37 C 10 min, 95 C 2 min, 50 C 30 s, 65 C 5 min, hold at 4 C 100 μl VOLUME 25 μl Program 11 Targeted Depletion 60 C 30 min, 95 C 5 min, hold at 4 C 50 μl AMPLIFICATION (FOR OPTIONAL ANYDEPLETE WORKFLOWS) VOLUME Program 12 Library Amplification B 95 C 2 min, 2x(95 C 30 s, 60 C 90 s), 15x* (95 C 30 s, 65 C 90 s), 65 C 5 min, hold at 4 C 100 μl Important Note: The number of cycles used for Library Amplification depends on the starting amount and quality of RNA. For more information, contact NuGEN Technical Support. Page 15 of 37 M01442 v2

20 V. Protocol This protocol includes workflows for RNA-Seq library construction with and without AnyDeplete. For standard workflows, follow sections A. Poly(A) Selection through J. Library Amplification A, then continue to N. Amplified Library Purification. For AnyDeplete workflows, follow sections A. Poly(A) Selection through I. Strand Selection Purification, then continue to K. Probe Binding (optional AnyDeplete workflow). For each section of the protocol, remove reagents from storage as listed. Thaw and place reagents at room temperature or on ice as instructed. After each section, continue immediately to the next section of the protocol unless otherwise directed. Return all reagents to their appropriate storage conditions promptly after use unless otherwise instructed. A. Poly(A) Selection Table 6. Oligo dt Bead Master Mix REAGENT OLIGO dt BEADS (CLEAR) mrna BINDING BUFFER (CLEAR) STORAGE 4 C 4 C 1X REACTION VOLUME 10 µl 50 µl 1. Remove Nuclease-free water (Green: D1) from -20 C and place at room temperature. Remove mrna Binding Buffer (Clear), mrna Wash Buffer (Clear), mrna Elution Buffer (Clear), and Oligo dt Beads (Clear) from 4 C storage and place at room temperature for 10 minutes prior to use. Ensure these reagents have completely reached room temperature before proceeding. 2. Prepare total RNA by diluting it with D1 to a final volume of 50 µl in a 0.2 ml tube and place on ice. 3. Prepare Bead Master Mix by combining mrna Binding Buffer and Oligo dt Beads in a in a 0.5 ml capped tube according to the volumes shown in Table Add 60 µl of Oligo dt Bead Master Mix to a 0.2 ml tube. 5. Add 50 µl of total RNA to 60 µl of Oligo dt Bead Master Mix. Mix thoroughly by pipetting up and down at least 10 times. Take care when pipetting to minimize foaming. 6. Place the tubes in a pre-warmed thermal cycler programmed to run Program 1 (Poly(A) RNA Binding; see Table 5): 65 C 5 min, hold at 4 C Page 16 of 37 M01442 v2

21 V. Protocol 7. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. 8. Mix samples thoroughly by pipetting up and down slowly at least 10 times. 9. Incubate at room temperature for 5 minutes. 10. Repeat steps 8 and 9 for a total of two incubations. 11. Transfer the tubes to the magnet and let stand for 2 minutes to completely clear the solution of beads. 12. Carefully remove the binding buffer and discard it, taking care not to disturb the beads. 13. Remove samples from the magnet. 14. Add 200 µl of mrna Wash Buffer to the beads. Mix thoroughly by slowly pipetting up and down at least 10 times. 15. Transfer the tubes to the magnet and let stand for 2 minutes to completely clear the solution of beads. 16. Carefully remove the wash buffer and discard it, taking care not to disturb the beads. 17. Remove tubes from the magnet. Remove 2x Fragmentation Buffer (Clear: FB1) from 20 C for use in the next step. Thaw at room temperature, mix by vortexing, spin down and place on ice. 18. Add 50 µl of mrna Elution Buffer to each sample. 19. Place the tubes in a pre-warmed thermal cycler programmed to run Program 2 (RNA Elution; see Table 5): 80 C 2 min, hold at 25 C 20. Remove tubes from the thermal cycler, spin to collect condensation and place on ice. 21. Add 50 µl of mrna Binding Buffer to bead/elution buffer mix. Incubate at room temperature 5 minutes. 22. Transfer the tubes to the magnet and let stand for 2 minutes to completely clear the solution of beads. 23. Carefully remove the buffer mix and discard it, taking care not to disturb the beads. 24. Remove the tubes from the magnet. 25. Add 200 µl of mrna Wash Buffer to the beads. Mix thoroughly by slowly pipetting up and down at least 10 times. 26. Transfer the tubes to the magnet and let stand for 2 minutes to completely clear the solution of beads. 27. Continue immediately to RNA Fragmentation. Page 17 of 37 M01442 v2

22 V. Protocol B. RNA Fragmentation Table 7. 1X Fragmentation Buffer REAGENT 2X FRAGMENTATION BUFFER (CLEAR: FB1 ver 1) NUCLEASE-FREE WATER (GREEN: D1) STORAGE 20 C 20 C 1X REACTION VOLUME 10 µl 10 µl 1. Prepare 1X Fragmentation Buffer by combining FB1 and D1 in a 0.5 ml capped tube according to the volumes shown in Table Carefully remove the wash buffer from the samples and discard it, taking care not to disturb the beads. 3. Remove the tubes from the magnet. 4. Resuspend the beads in 20 µl of 1X Fragmentation Buffer. 5. Place the tubes in a pre-warmed thermal cycler programmed to run Program 3 (RNA Fragmentation; see Table 5): 94 C 8 min, hold at 4 C 6. Remove tubes from the thermal cycler, spin to collect condensation and place on ice. 7. Transfer the tubes to the magnet and let stand for 2 minutes to completely clear the solution of beads. 8. Transfer 20 µl of fragmented mrna to a new 0.2 ml tube and place on ice. C. First Strand cdna Synthesis Table 8. First Strand Master Mix REAGENT ACTINOMYCIN D (BROWN) FIRST STRAND BUFFER MIX (BLUE: A2 ver 14) FIRST STRAND ENZYME MIX (BLUE: A3 ver 6) STORAGE 20 C 20 C 20 C 1X REACTION VOLUME 1.25 µl 2.75 µl 1 µl 1. Spin down the contents of A3 and place on ice. Page 18 of 37 M01442 v2

23 V. Protocol 2. Thaw Actinomycin D and A2 at room temperature. Mix by vortexing, spin down and place on ice. 3. Prepare a master mix by combining Actinomycin D, A2 and A3 in a 0.5 ml capped tube according to the volumes shown in Table 8. Mix well by pipetting, spin down, and place on ice. 4. Add 5 µl of First Strand Master Mix to each sample tube for a total of 25 µl. Mix well by pipetting, spin down and place on ice. 5. Place the tubes in a thermal cycler programmed to run Program 4 (First Strand Synthesis; see Table 5): 25 C 5 min, 42 C 15 min, 70 C 15 min, hold at 4 C 6. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. D. Second Strand cdna Synthesis Table 9. Second Strand Master Mix REAGENT SECOND STRAND BUFFER MIX (YELLOW: B1 ver 11) SECOND STRAND ENZYME MIX (YELLOW: B2 ver 4) STORAGE 20 C 20 C 1X REACTION VOLUME 48 µl 2 µl 1. Remove Agencourt beads from 4 C storage and Nuclease-free water (Green: D1) from -20 C storage and place on the bench top to reach room temperature for use in the next step. 2. Spin down the contents of B2 and place on ice. 3. Thaw B1 at room temperature, mix by vortexing, spin down and place on ice. 4. Prepare a master mix by combining B1 and B2 in a 0.5 ml capped tube according to the volumes shown in Table 9. Mix well by pipetting, spin down, and place on ice. 5. Add 50 µl of Second Strand Master Mix to each sample tube for a total of 75 µl. Mix well by pipetting, spin down and place on ice. Page 19 of 37 M01442 v2

24 V. Protocol 6. Place the tubes in a pre-chilled thermal cycler programmed to run Program 5 (Second Strand Synthesis; see Table 5): 16 C 60 min, hold at 4 C 7. Remove the tubes from the thermal cycler and spin to collect condensation. E. cdna Purification 1. Ensure the Agencourt beads and D1 have completely reached room temperature before proceeding. Prepare 70% ethanol daily using fresh ethanol stocks. 2. Prepare a 70% ethanol wash solution. It is critical that this solution be prepared fresh on the same day of the experiment from a recently opened stock container. Note: Measure both the ethanol and the water carefully prior to mixing. Failure to do so can result in a higher than anticipated aqueous content, which may reduce amplification yield. 3. Resuspend the beads by inverting and tapping the tube. Ensure that the beads are fully resuspended before adding to the sample. After resuspending, do not spin the beads. 4. At room temperature, add 135 µl (1.8 volumes) of Agencourt beads to each reaction and mix by pipetting 10 times. Note: At this step, the reaction will be 210 µl. Pipet carefully to avoid spilling the sample. 5. Incubate at room temperature for 10 minutes. 6. Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads. 7. Keeping the tubes on the magnet, carefully remove the binding buffer and discard it. 8. With the tubes still on the magnet, add 200 µl of freshly prepared 70% ethanol and allow to stand for 30 seconds. Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact cdna yields, so ensure beads are not removed with the binding buffer or the washes. 9. Remove the 70% ethanol wash using a pipette. Note: It is critical to remove as much of the ethanol as possible. Use at least 2 pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. 10. Air-dry the beads on the magnet for 10 minutes. Inspect each tube carefully to ensure that all the ethanol has evaporated. 11. Remove the tubes from the magnet. Page 20 of 37 M01442 v2

25 V. Protocol 12. Add 11 µl D1 to the dried beads. Mix thoroughly to ensure all beads are resuspended. 13. Incubate at room temperature for 5 minutes. 14. Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads. 15. Transfer 10 µl of sample to a fresh 0.2 ml tube. Optional stopping point: Store samples at 20 C. F. End Repair Table 10. End Repair Master Mix REAGENT END REPAIR BUFFER MIX (BLUE: ER1 ver 7) END REPAIR ENZYME MIX (BLUE: ER2 ver 4) END REPAIR ENHANCER (BLUE: ER3 ver 2) STORAGE 20 C 20 C 20 C 1X REACTION VOLUME 4 µl 0.5 µl 0.5 µl 1. Spin down the contents of ER2 and ER3 and place on ice. 2. Thaw ER1 at room temperature. Mix by vortexing, spin down and place on ice. 3. Prepare a master mix by combining ER1, ER2 and ER3 in a 0.5 ml capped tube according to the volumes shown in Table 10. Mix well by pipetting, spin down and place on ice. 4. Add 5 µl of End Repair Master Mix to each sample tube for a total of 15 μl. Mix by pipetting, spin down and place on ice. 5. Place the tubes in a thermal cycler programmed to run Program 6 (End Repair; see Table 5): 25 C 30 min, 70 C 10 min, hold at 4 C 6. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. Page 21 of 37 M01442 v2

26 V. Protocol G. Adaptor Ligation Table 11. Ligation Master Mix REAGENT NUCLEASE- FREE WATER (GREEN: D1) LIGATION BUFFER MIX (YELLOW: L1 ver 4) LIGATION ENZYME MIX (YELLOW: L3 ver 4) STORAGE 20 C 20 C 20 C 1X REACTION VOLUME 4.5 µl 6.0 µl 1.5 µl 1. Spin down L3 and place on ice. 2. Thaw Adaptor Mixes (L2V22DR-BC) or Adaptor Plate on ice, spin down, and return to ice. 3. Thaw L1 at room temperature. Mix by vortexing, spin down and place on ice. 4. Add 3 µl of the appropriate barcoded Adaptor Mix to each sample. Mix thoroughly by pipetting, spin down and place on ice. Make sure a unique barcode is used for each sample to be multiplexed together on the sequencer. 5. Prepare a master mix by combining D1, L1 and L3 in a 0.5 ml capped tube according to the volumes shown in Table 11. Mix by pipetting slowly, without introducing bubbles, spin down and place on ice. Use the master mix immediately. Note: The L1 Ligation Buffer Mix is very viscous. Please be sure to pipet this reagent slowly. 6. Add 12 µl of Ligation Master Mix to each reaction tube for a total of 30 µl. Mix thoroughly by pipetting slowly and gently, spin down and place on ice. Proceed immediately with the incubation. 7. Place the tubes in a thermal cycler programmed to run Program 7 (Adaptor Ligation; see Table 5): 25 C 30 min, hold at 4 C 8. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. Page 22 of 37 M01442 v2

27 V. Protocol H. Strand Selection Table 12. Strand Selection Master Mix REAGENT STRAND SELECTION BUFFER MIX I (PURPLE: SS1) STRAND SELECTION ENZYME MIX I (PURPLE: SS2) STORAGE 20 C 20 C 1X REACTION VOLUME 69 µl 1 µl 1. Remove Agencourt beads from 4 C storage and Nuclease-free water (Green: D1) from -20 C storage and place on the bench top to reach room temperature for use in the next step. 2. Thaw SS1 at room temperature. Mix by vortexing, spin down and place on ice. 3. Spin down SS2 and place on ice. 4. Prepare a master mix by combining SS1 and SS2 in a 0.5 ml capped tube according to the volumes shown in Table Add 70 µl of Strand Selection Master Mix to 30 µl of each sample for a total of 100 µl. Mix by pipetting, spin down and place on ice. 6. Place the tubes in a pre-warmed thermal cycler programmed to run Program 8 (Strand Selection; see Table 5): 72 C 10 min, hold at 4 C 7. Remove the tubes from the thermal cycler and spin to collect condensation. I. Strand Selection Purification 1. Ensure the Agencourt beads and D1 have completely reached room temperature before proceeding. 2. Resuspend the beads by inverting and tapping the tube. Ensure the beads are fully resuspended before adding to samples. After resuspending, do not spin the beads. 3. Add 80 µl (0.8 volumes) of the bead suspension to the Strand Selection reaction product. Mix thoroughly by pipetting up and down. 4. Incubate at room temperature for 10 minutes. 5. Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads. 6. Carefully remove 165 µl of the binding buffer and discard it. Leaving some of the volume behind minimizes bead loss at this step. Page 23 of 37 M01442 v2

28 V. Protocol Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact the amount of DNA carried into subsequent steps of the protocol, so ensure beads are not removed with the binding buffer or the wash. 7. With the tubes still on the magnet, add 200 µl of freshly prepared 70% ethanol and allow to stand for 30 seconds. 8. Remove the 70% ethanol wash using a pipette. Note: It is critical to remove as much of the ethanol as possible. Use at least two pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. 9. Air-dry the the beads on the magnet for 10 minutes. Inspect each tube carefully to ensure that all of the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing. 10. Remove the tubes from the magnet. 11. Add 16 µl D1 to the dried beads. Mix thoroughly to ensure all beads are resuspended. 12. Transfer the tubes to the magnet and let stand for 3 minutes for the beads to clear the solution. 13. Carefully remove 15 µl of the eluate, ensuring as few beads as possible are carried over, transfer to a fresh set of PCR tubes and place on ice. 14. Continue to Section J. Library Amplification A (for standard workflows) or to Section K. Probe Binding (for optional AnyDeplete workflows). Optional stopping point: Store samples at 20 C. J. Library Amplification A Table 13. Library Amplification A Master Mix REAGENT AMPLIFICATION BUFFER MIX (RED: P1 ver 4) AMPLIFICATION PRIMER MIX (RED: P2 ver 10) AMPLIFICATION ENZYME MIX (RED: P3 ver 2) STRAND SELECTION ENZYME MIX II (PURPLE: SS4) NUCLEASE- FREE WATER (GREEN: D1) STORAGE 20 C 20 C 20 C 20 C 20 C 1X REACTION VOLUME 10 µl 8 µl 0.5 µl 1 µl 65.5 µl Page 24 of 37 M01442 v2

29 V. Protocol 1. Remove Agencourt beads from 4 C storage and Nuclease-free water (Green: D1) from -20 C storage and place on the bench top to reach room temperature for use in the next step. 2. Thaw P1 and P2 at room temperature. Mix by vortexing, spin down and place on ice. 3. Spin down P3 and SS4 and place on ice. 4. Make a master mix by sequentially combining D1, P1 and P2 in an appropriately sized capped tube according to the volumes shown in Table 13. Add P3 and SS4 at the last moment and mix well by pipetting, taking care to avoid bubbles. Spin down and place on ice. 5. On ice, add 85 µl of Library Amplification A Master Mix to each sample for a total of 100 µl. Mix by pipetting, spin down and place on ice Place the tubes in a pre-warmed thermal cycler programmed to run Program 9 (Library Amplification A; see Table 5): 37 C 10 min, 95 C 2 min, 2x(95 C 30 s, 60 C 90 s), 15x* (95 C 30 s, 65 C 90 s), 65 C 5 min, hold at 4 C *The precise number of PCR cycles required depends on a number of factors including sample type, quality and input amount. The number of PCR cycles may be decreased or increased based on the requirements for a given sample and should be optimized via qpcr. Contact techserv@nugen.com for more information. 7. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. 8. Continue to N. Amplified Library Purification. Optional stopping point: Store samples at 20 C. Page 25 of 37 M01442 v2

30 V. Protocol K. Probe Binding (optional AnyDeplete workflow) Table 14. Probe Binding Master Mix REAGENT ANYDEPLETE BUFFER MIX (PURPLE: AD1 ver 1) ANYDEPLETE PROBE MIX (PURPLE: ADXX or ICXXXX) ANYDEPLETE ENZYME MIX I (PURPLE: AD2 ver 1) STRAND SELECTION ENZYME MIX II (PURPLE: SS4) STORAGE 20 C 20 C 20 C 20 C 1X REACTION VOLUME 5 µl 4 µl 0.5 µl 0.5 µl 1. Thaw AD1 and ADXX or custom AnyDeplete Probe Mix at room temperature. Mix by pipetting, spin down and place on ice. 2. Spin down AD2 and SS4 and place on ice. 3. Prepare a master mix by combining AD1, ADXX or custom AnyDeplete Probe Mix, AD2 and SS4 in a 0.5 ml capped tube, according to the volumes shown in Table Add 10 µl of Probe Binding Master Mix to each sample for a total of 25 µl. Mix by pipetting, spin down and place on ice. 5. Place the tubes in a pre-warmed thermal cycler programmed to run Program 10 (Probe Binding; see Table 5): 37 C 10 min, 95 C 2 min, 50 C 30 s, 65 C 5 min, hold at 4 C 6. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. L. Targeted Depletion (optional AnyDeplete workflow) Table 15. Targeted Depletion Master Mix REAGENT ANYDEPLETE BUFFER MIX (PURPLE: AD1 ver 1) ANYDEPLETE ENZYME MIX II (PURPLE: AD3 ver 1) NUCLEASE-FREE WATER (GREEN: D1) STORAGE 20 C 20 C 20 C 1X REACTION VOLUME 5 µl 4 µl 16 µl Page 26 of 37 M01442 v2

31 V. Protocol 1. Thaw D1 and AD1 at room temperature. Mix AD1 by vortexing, spin down and place on ice. 2. Spin down AD3 and place on ice. 3. Prepare a master mix by combining AD1, AD3 and D1 in a 0.5 ml capped tube according the volumes in Table 15. Mix thoroughly by pipetting, spin down and place on ice. 4. Add 25 µl of Targeted Depletion Master Mix to each sample for a total of 50 µl. Mix by pipetting, spin down and place on ice. 5. Place the tubes in a pre-warmed thermal cycler programmed to run Program 11 (Targeted Depletion; see Table 5): 60 C 30 min, 95 C 5 min, hold at 4 C 6. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. M. Library Amplification B (optional AnyDeplete workflow) Note: The AnyDeplete workflow eliminates unwanted reads from the library. Depending on the starting concentration of unwanted reads in the library and the final library concentration after AnyDeplete, additional PCR cycles may be needed to obtain sufficient yield for sequencing. For additional information contact NuGEN Technical Support. Table 16. Library Amplification B Master Mix REAGENT AMPLIFICATION BUFFER MIX (RED: P1 ver 4) AMPLIFICATION PRIMER MIX (RED: P2 ver 10) AMPLIFICATION ENZYME MIX (RED: P3 ver 2) NUCLEASE- FREE WATER (GREEN: D1) STORAGE 20 C 20 C 20 C 20 C 1X REACTION VOLUME 10 µl 8 µl 0.5 µl 31.5 µl 1. Remove Agencourt beads from 4 C storage and Nuclease-free water (Green: D1) from -20 C storage and place on the bench top to reach room temperature for use in the next step. Page 27 of 37 M01442 v2

32 V. Protocol 2. Thaw P1 and P2 at room temperature. Mix by vortexing, spin down and place on ice. 3. Spin down P3 and place on ice. 4. Prepare a master mix by sequentially combining D1, P1 and P2 in an appropriately sized capped tube according to the volumes shown in Table 16. Add P3 at the last moment and mix well by pipetting, taking care to avoid bubbles. Spin down and place on ice. 5. On ice, add 50 μl of Library Amplification B Master Mix to each sample for a total of 100 μl. Mix by pipetting, spin down and place on ice. 6. Place the tubes in a pre-warmed thermal cycler programmed to run Program 12 (Library Amplification B; see Table 5): 95 C 2 min, 2x(95 C 30 s, 60 C 90 s), 17x*(95 C 30 s, 65 C 90 s), 65 C 5 min, hold at 4 C *The precise number of PCR cycles required depends on a number of factors including sample type, quality and input amount. The number of PCR cycles may be decreased or increased based on the requirements for a given sample and should be optimized via qpcr. Contact techserv@nugen.com for more information. The recommended number of PCR cycles is based on starting with a 100 ng of total RNA from whole blood containing approximately 30% globin, and performing globin AnyDeplete. 7. Remove the tubes from the thermal cycler, spin to collect condensation and place on ice. Optional stopping point: Store samples at 20 C. N. Amplified Library Purification 1. Ensure the Agencourt beads and D1 have completely reached room temperature before proceeding. 2. Resuspend the beads by inverting and tapping the tube. Ensure the beads are fully resuspended before adding to samples. After resuspending, do not spin the beads. 3. At room temperature, add 100 μl of the Library Amplification B reaction product to 100 μl (1.0 volumes) of bead suspension. Mix thoroughly by pipetting up and down. Note: At this step, the reaction will be 200 μl. Pipet carefully to avoid spilling the sample. Page 28 of 37 M01442 v2

33 V. Protocol 4. Incubate at room temperature for 10 minutes. 5. Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads. 6. Carefully remove 200 μl of the binding buffer and discard it. Note: The beads should not disperse; instead, they will stay on the walls of the tubes. Significant loss of beads at this stage will impact the amount of final library yield, so ensure beads are not removed with the binding buffer or the wash. 7. With the tubes still on the magnet, add 200 μl of freshly prepared 70% ethanol and allow to stand for 30 seconds. 8. Remove the 70% ethanol wash using a pipette. 9. Repeat the 70% ethanol wash one more time, for a total of two washes. Note: With the final wash, it is critical to remove as much of the ethanol as possible. Use at least two pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step. 10. Air dry the beads on the magnet for 10 minutes. Inspect each tube carefully to ensure that all of the ethanol has evaporated. It is critical that all residual ethanol be removed prior to continuing. 11. Remove the tubes from the magnet. 12. Add 31 μl D1 to the dried beads. Mix thoroughly to ensure all beads are resuspended. 13. Transfer the tubes to the magnet and let stand for 3 minutes for the beads to clear the solution. 14. Carefully remove 30 μl of the eluate, ensuring as few beads as possible are carried over, transfer to a fresh set of PCR tubes and place on ice. Optional stopping point: Store samples at 20 C. O. Quantitative and Qualitative Assessment of the Library 1. Assess the library by running 1 μl of 5 ng/μl library on the High Sensitivity DNA Chip (Agilent Technologies). A typical fragment distribution for high quality inputs is shown in Figure 4. Page 29 of 37 M01442 v2

34 V. Protocol Figure 4. Fragment distribution when 1 μl of 5 ng/μl library is loaded into a High Sensitivity DNA Chip from 100 ng K562 total RNA starting material. 2. Quantify the library using a qpcr-based method. 3. Validate the library as described in Illumina User Guides for DNA library construction, e.g., Genomic DNA Sample Prep Manual (Cat. #FC ). Page 30 of 37 M01442 v2

35 VI. Technical Support For help with any of our products, please contact NuGEN Technical Support at (direct) or , option 2 (toll-free, U.S. only). You may also send faxes to (toll-free) or techserv@nugen.com. In Europe contact NuGEN at +31(0) (Phone) or +31(0) (Fax) or europe@nugen.com. In all other locations, contact your NuGEN distributor for technical support. Page 31 of 37 M01442 v2

36 VII. Appendix A. Barcode Sequences and Guidelines for Multiplex Experiments Barcode sequences for the Adaptor Plates are given below. Barcodes 1 8 in the 8-reaction kit correspond to plate positions A01 H01, respectively. Barcodes in the 32 reaction kits are found in wells A01 H04, respectively. Barcodes are color balanced in pairs (i.e. A01 + B01, C01 + D01, etc.) and in sets of 8 by column. Table 17. Barcode sequences for adaptors PLATE POSITION BARCODE SEQUENCE PLATE POSITION BARCODE SEQUENCE PLATE POSITION BARCODE SEQUENCE A01 CGCTACAT A05 AGGTTCCT A09 GCCTTAAC B01 AATCCAGC B05 GAACCTTC B09 ATTCCGCT C01 CGTCTAAC C05 AAGTCCTC C09 ATCGTGGT D01 AACTCGGA D05 CCACAACA D09 GCTACAAC E01 GTCGAGAA E05 ATAACGCC E09 TCTACGCA F01 ACAACAGC F05 CCGGAATA F09 CTCCAATC G01 ATGACAGG G05 CCAAGTAG G09 ACTCTCCA H01 GCACACAA H05 AAGGACCA H09 GTCTCATC A02 CTCCTAGT A06 ACGCTTCT A10 GCCAGAAT B02 TCTTCGAC B06 CTATCCAC B10 AATGACGC C02 GACTACGA C06 TGACAACC C10 GTACCACA D02 ACTCCTAC D06 CAGTGCTT D10 ACGATCAG E02 CTTCCTTC E06 TCACTCGA E10 TAACGTCG F02 ACCATCCT F06 CTGACTAC F10 CGCAACTA G02 CGTCCATT G06 GTGATCCA G10 AACACTGG H02 AACTTGCC H06 ACAGCAAG H10 CCTGTCAA A03 GTACACCT A07 TGCTGTGA A11 TCCTGGTA B03 ACGAGAAC B07 CAACACAG B11 CATCAACC C03 CGACCTAA C07 CCACATTG C11 AGCAGACA D03 TACATCGG D07 TAGTGCCA D11 GAAGACTG E03 ATCGTCTC E07 TCGTGCAT E11 TCTAGTCC F03 CCAACACT F07 CTACATCC F11 CTCGACTT G03 TCTAGGAG G07 CATACGGA G11 CTAGCTCA H03 CTCGAACA H07 TGCGTAAC H11 TCCAACTG A04 ACGGACTT A08 CAGGTTCA A12 GACATCTC B04 CTAAGACC B08 AGAACCAG B12 ACTGCACT C04 AACCGAAC C08 GAATGGCA C12 GTTCCATG D04 CCTTAGGT D08 AGGCAATG D12 ACCAAGCA E04 CCTATACC E08 TAGGAGCT E12 CTCTCAGA F04 AACGCCTT F08 CGAACAAC F12 ACTCTGAG G04 TCCATTGC G08 CATTCGTC G12 GCTCAGTT H04 CAAGCCAA H08 AGCCAACT H12 ATCTGACC Page 32 of 37 M01442 v2