mRNA-Seq_SamplePrep.book

mrna Sequencing Sample Preparation Guide Topics 3 Introduction 4 RNA Input Recommendations 6 mrna-seq Sample Preparation Kit Contents 8 User-Supplied Consumables and Equipment 9 Purify the mrna 12 Fragment the mrna 14 Synthesize the First Strand cdna 15 Synthesize the Second Strand cdna 16 Perform End Repair 17 Adenylate 3' Ends 18 Ligate the Adapters 19 Purify the cdna Templates 21 Enrich the Purified cdna Templates 22 Validate the Library ILLUMINA PROPRIETARY Cat # RS-930-1001 Part # 1004898 Rev. D September 2009

Illumina, Inc. Illumina Illumina, Inc. Illumina Illumina Illumina Illumina Illumina Illumina Illumina Illumina Illumina 2009 Illumina, Inc. All rights reserved. Illumina illuminadx Solexa Making Sense Out of Life Oligator Sentrix GoldenGate GoldenGate Indexing DASL BeadArray Array of Arrays Infinium BeadXpress VeraCode IntelliHyb iselect CSPro GenomeStudio Illumina, Inc. Phusion Finnzymes Oy DNA New England Biolabs, Inc. 5,500,363 5,352,778 BIO-RAD Laboratories, Inc.

3 Introduction total RNA DNA 1 poly-t poly- A mrna mrna RNA 1st strand cdna DNA I RNaseH 2nd strand cdna cdna A PCR cdna Sample Prep Workflow 2 Day 1 Day 2 Start with 1 10 μg total RNA Repair Ends Purify and Fragment mrna Add A Bases to 3 Ends First Strand cdna Synthesis Ligate Adapters Second Strand cdna Synthesis Purify Ligation Product Cold Storage Option PCR Amplification Figure 1 mrna-seq Sample Preparation Workflow mrna Sample Preparation Guide

4 RNA Input Recommendations Best Practices Starting Material RNA RNase RNase RNase RNA total RNA RNA RNA RNase RNase RNase RNase RNA RNA RNA DNA RNase/DNase Total RNA Input total RNA 1 10 μg Universal Human Reference total RNA 1 μg RNA RNA RNA RNA 5' total RNA Agilent Technologies 2100 Bioanalyzer RNA Integrity Number RIN 8 1% RNA RNA 28S rrna 4.5 kb 18S rrna 1.9 kb 28S 18S 2 RNA 6000 ladder mrna 0.5 12 kb Universal Human Reference UHR RNA Bioanalyzer Figure 2 Cat # RS-930-1001 Part # 1004898 Rev. D

5 Figure 2 Starting RNA Bioanalyzer Trace Purified mrna Input mrna total RNA 1 10 μg mrna mrna mrna Positive Control Stratagene Human UHR total RNA catalog # 740000 mrna Sample Preparation Guide

6 mrna-seq Sample Preparation Kit Contents Kit Contents, Box 1 Store at -15 to -25 C Box 1-15 -25ºC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Figure 3 mrna-seq Sample Prep Kit, Box 1, part # 1004814 1. Ultra Pure Water, part # 1001913 2. 10 mm Tris Buffer, part # 1002115 3. 5X Fragmentation Buffer, part # 1005084 4. Fragmentation Stop Solution, part # 1004826 5. Glycogen, part # 1001664 6. Random Primers, part # 1004784 7. 25 mm dntps Mix, part # 11318102 8. RNase Inhibitor, part # 15003548 9. GEX Second Strand Buffer, part # 1000562 10. RNaseH, part # 1000576 11. DNA Polymerase I, part # 1000577 12. 10X End Repair Buffer, part # 1004819 13. T4 DNA Polymerase, part # 1000514 14. Klenow DNA Polymerase, part # 1000515 15. T4 PNK, part # 1005082 Cat # RS-930-1001 Part # 1004898 Rev. D

7 16. 10X A-Tailing Buffer, part # 1002105 17. 1 mm datp, part # 11318081 18. Klenow Exo-, part # 11318090 19. 2X Rapid T4 DNA Ligase Buffer, part # 1004792 20. PE Adapter Oligo Mix, part # 1001782 21. T4 DNA Ligase, part # 1004790 22. 5X Phusion Buffer (Finnzymes Oy), part # 1000585 23. PCR Primer PE 2.0, part # 1001784 24. PCR Primer PE 1.0, part # 1001783 25. Phusion DNA Polymerase (Finnzymes Oy), part # 1000584 26. 27. 28. 29. Bead Binding Buffer, part # 1002118 30. Bead Washing Buffer, part # 1004800 NOTE Kit Contents, Bag 1 Store at 2 to 8 C Bag 1 part # 1004815 4ºC 2 8ºC Sera-Mag Magnetic Oligo(dT) Beads, part # 1004815 mrna Sample Preparation Guide

8 User-Supplied Consumables and Equipment Table 1 User-Supplied Consumables Consumable Supplier 1.5 ml RNAse-free non-sticky tube Ambion, part # AM12450 3 M NaOAc, ph 5.2 General lab supplier 50x TAE Buffer General lab supplier 70% EtOH General lab supplier 100% EtOH General lab supplier 100 bp DNA Ladder Invitrogen, part # 10488-058 6X DNA loading dye General lab supplier Certified Low-Range Ultra Agarose BIO-RAD, part # 161-3106 GeneCatcher Disposable Gel Excision Kit Gel Company, part # PKB6.5 MinElute PCR Purification Kit QIAGEN, part # 28004 QIAquick Gel Extraction Kit QIAGEN, part # 28704 QIAquick PCR Purification Kit QIAGEN, part # 28104 SuperScript II Reverse Transcriptase with 100 mm DTT and 5X First Strand Buffer Invitrogen, part # 18064-014 Table 2 Equipment Checklist Consumable Supplier 4 C microcentrifuge General lab supplier Heat block (2) Magnetic stand Room temperature tube rotator Thermal cycler General lab supplier General lab supplier General lab supplier General lab supplier Cat # RS-930-1001 Part # 1004898 Rev. D

9 Purify the mrna poly-t poly-a mrna Consumables Best Practice Illumina-Supplied Sera-Mag Magnetic Oligo(dT) Beads Bead Binding Buffer Bead Washing Buffer Ultra Pure Water 10 mm Tris Buffer Using the Magnetic Stand NOTE 1. 1 2 CAUTION 2. 3. 0.5 1 CAUTION 4. 1 2 5. 1 4 mrna Sample Preparation Guide

10 Procedure 1 2 NOTE total RNA 1 10 μg NOTE mrna 100 ng Fragment the mrna 12 1. 1 65ºC 1 80ºC 2. 1.5 ml RNase total RNA 50 μl 3. 65ºC 5 4. 1.5 ml RNase Sera-mag Magnetic Oligo(dT) Beads 15 μl 5. Bead Binding Buffer 100 μl 2 6. Bead Binding Buffer 50 μl 3 total RNA 50 μl 7. 6 5 8. 1.5 ml RNase Bead Binding Buffer 50 μl 9. 5 7 Bead Washing Buffer 200 μl 2 10. 10 mm Tris-HCl 50 μl 80ºC 2 mrna 11. mrna 8 12. Bead Washing Buffer 200 μl 2 13. 65ºC 5 14. 13 mrna 100 μl 5 15. Bead Washing Buffer 200 μl 2 Cat # RS-930-1001 Part # 1004898 Rev. D

11 16. 10 mm Tris-HCl 17 μl 80ºC 2 mrna 17. mrna 200 μl thin-wall PCR mrna 16 μl mrna Sample Preparation Guide

12 Fragment the mrna mrna Consumables Illumina-Supplied 5X Fragmentation Buffer Fragmentation Stop Solution Glycogen Ultra Pure Water User-Supplied 3 M NaOAC, ph 5.2 70% EtOH 100% EtOH Procedure 1. PCR 94ºC 2. 200 μl thin-wall PCR 5X Fragmentation Buffer (4 μl) mrna (16 μl) 20 μl 3. 94ºC PCR 5 4. Fragmentation Stop Solution 2 μl 5. 6. 1.5 ml RNase 7. -80ºC 30 3 M NaOAC, ph 5.2 (2 μl) Glycogen (2 μl) 100% EtOH (60 μl) RNA -15-25ºC 8. 4ºC 14,000 rpm 20,200 g 25 9. RNA EtOH RNA EtOH 90% Cat # RS-930-1001 Part # 1004898 Rev. D

13 10. 70% EtOH 300 μl 11. 70% EtOH 12. 10 13. RNase 11.1 μl RNA mrna Sample Preparation Guide

14 Synthesize the First Strand cdna RNA 1st strand cdna Consumables Illumina-Supplied 25 mm dntp Mix Random Primers RNase Inhibitor User-Supplied SuperScript II 100 mm DTT SuperScript II 5X First Strand Buffer SuperScript II Procedure 1. 200 μl thin-wall PCR Random Primers (1 μl) mrna (11.1 μl) 12.1 μl 2. 65ºC PCR 5 3. PCR 25ºC 4. 10% 5X First Strand Buffer (4 μl) 100 mm DTT (2 μl) 25 mm dntp Mix (0.4 μl) RNase Inhibitor (0.5 μl) 6.9 μl 5. 6.9 μl PCR 6. 25ºC PCR 2 7. SuperScript II 1 μl a. 25ºC 10 b. 42ºC 50 c. 70ºC 15 d. 4ºC 8. Cat # RS-930-1001 Part # 1004898 Rev. D

15 Synthesize the Second Strand cdna RNA cdna Consumables Illumina-Supplied Ultra Pure Water GEX Second Strand Buffer 25 mm dntp Mix RNaseH DNA Pol I User-Supplied QIAquick PCR Purification Kit (QIAGEN, part # 28104) Procedure 1. PCR 16ºC 2. Ultra Pure Water 62.8 μl 1st strand cdna 3. GEX Second Strand Buffer (10 μl) 25 mm dntp Mix (1.2 μl) 4. 5 5. RNaseH (1 μl) DNA Pol I (5 μl) 6. 16ºC 2.5 7. QIAquick PCR Purification Kit 50 μl QIAGEN EB Buffer 8. DNA -15-25ºC mrna Sample Preparation Guide

16 Perform End Repair T4 DNA Klenow DNA 3' 5' 3' 5' Consumables Illumina-Supplied 10X End Repair Buffer 25 mm dntp Mix T4 DNA Polymerase Klenow DNA Polymerase T4 PNK Ultra Pure Water User-Supplied QIAquick PCR Purification Kit (QIAGEN, part # 28104) Procedure 1. 1 20ºC 1 37ºC 2. 1.5 ml RNase DNA (50 μl) Water (27.4 μl) 10X End Repair Buffer (10 μl) 25 mm dntp Mix (1.6 μl) T4 DNA Polymerase (5 μl) Klenow DNA Polymerase (1 μl) T4 PNK (5 μl) 100 μl 3. 20ºC 30 4. QIAquick PCR Purification Kit 32 μl QIAGEN EB Buffer -15-25ºC Cat # RS-930-1001 Part # 1004898 Rev. D

17 Adenylate 3' Ends Klenow 3' to 5' exo minus DNA 3' A 3' T DNA Consumables Illumina-Supplied A-Tailing Buffer 1 mm datp Klenow exo (3' to 5' exo minus) (3 μl) User-Supplied MinElute PCR Purification Kit (QIAGEN, part # 28004) NOTE QIAquick MinElute Procedure 1. 1.5 ml RNase DNA (32 μl) A-Tailing Buffer (5 μl) 1 mm datp (10 μl) Klenow exo (3' to 5' exo minus) (3 μl) 50 μl 2. 37ºC 30 3. MinElute PCR Purification Kit 23 μl QIAGEN EB Buffer -15-25ºC mrna Sample Preparation Guide

18 Ligate the Adapters DNA DNA NOTE mrna Consumables Illumina-Supplied 2X Rapid T4 DNA Ligase Buffer PE Adapter Oligo Mix T4 DNA Ligase User-Supplied MinElute PCR Purification Kit (QIAGEN, part # 28004) Procedure 1. 1.5 ml RNase DNA (23 μl) 2X Rapid T4 DNA Ligase Buffer (25 μl) PE Adapter Oligo Mix (1 μl) T4 DNA Ligase (1 μl) 50 μl 2. 15 3. MinElute PCR Purification Kit 10 μl QIAGEN EB Buffer NOTE QIAGEN PE 2-15 -25ºC Cat # RS-930-1001 Part # 1004898 Rev. D

19 Purify the cdna Templates Consumables User-Supplied Certified Low-Range Ultra Agarose 50 TAE 100 bp DNA Ladder 6 DNA GeneCatcher Disposable Gel Excision Kit QIAquick Gel Extraction Kit (QIAGEN, part # 28704) Procedure 1. TAE 2% 50 ml TAE 50 ml 1 2. 1 100 bp DNA Ladder 2 μl 2 DNA 10 μl 6 DNA 2 μl 3 100 bp DNA Ladder 2 μl NOTE 1 1 3 3. 120 V 60 4. 1 200 bp ±25 bp Figure 4 NOTE 500 bp ±25 bp NOTE DNA mrna Sample Preparation Guide

20 300 bp 200 bp (Cut-out) 100 bp Figure 4 2% Agarose Gel 5. QIAquick Gel Extraction Kit 30 μl QIAGEN EB Buffer -15-25ºC Cat # RS-930-1001 Part # 1004898 Rev. D

21 Enrich the Purified cdna Templates PCR cdna 2 PCR Consumables Illumina-Supplied 5X Phusion Buffer (Finnzymes Oy) Phusion DNA Polymerase (Finnzymes Oy) PCR Primer PE 1.0 PCR Primer PE 2.0 25 mm dntp Mix Ultra Pure Water User-Supplied QIAquick PCR Purification Kit (QIAGEN, part # 28104) Procedure 1. 200 μl thin-wall PCR PCR 10% 5X Phusion Buffer (10 μl) PCR Primer PE 1.0 (1 μl) PCR Primer PE 2.0 (1 μl) 25 mm dntp Mix (0.5 μl) Phusion DNA Polymerase (0.5 μl) Water (7 μl) 20 μl 2. 200 μl PCR 5 30 μl 3. PCR a. 98ºC 30 b. 15 98ºC 10 65ºC 30 72ºC 30 c. 72ºC 5 d. 4ºC 4. QIAquick PCR Purification Kit 30 μl QIAGEN EB Buffer -15-25ºC mrna Sample Preparation Guide

22 Validate the Library DNA 1. 1 μl Agilent Technologies 2100 Bioanalyzer Agilent DNA-1000 DNA Figure 5 Final mrna-seq Library Bioanalyzer Trace 2. 200 bp Figure 6 200 bp PCR Product 1 μl Invitrogen Zero Blunt TOPO Cat # RS-930-1001 Part # 1004898 Rev. D

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