Illumina TruSeq Stranded mrna (LT) Protocol 1

Transcription

1 Illumina TruSeq Stranded mrna (LT) Protocol 1 Performed using the TruSeq Stranded mrna Sample Preparation Kit (A cat#fc , B cat#fc ) Purify and Fragment mrna NOTE: Use 500ng of Total RNA to initiate protocol ( ug starting range recommended or ng of isolated mrna by illumina). See original protocol for use of purified mrna. NOTE: Thaw frozen Bead Binding Buffer (BBB), Bead Washing Buffer (BWB), Elution Buffer (ELB) and Fragment, Prime, Finish Mix (FPF) at RT. Store BBB, BWB, ELB and Resuspension Buffer at 4 C for subsequent experiments. NOTE: Remove RNA Purification Beads from storage and let stand at RT. NOTE: Pre-Chill 96 well plate centrifuge and plate adapter to 15 C. 1. Dilute 500ng of total RNA to 50ul using nuclease free H2O in a new 96 well 0.3ml non-skirted PCR plate. Apply RNA Bead Plate (RBP) Barcode label to plate. 2. Vortex the warmed RNA Purification Beads (RPB) vigorously to completely resuspend the Oligo-dT beads. 3. Add 50ul of the RPB to each well of the plate. Gently pipette up and down 6xs to mix beads. 4. Seal plate with adhesive plate seal and incubate in thermal cycler using the following profile to denature RNA : RNADNT 65 C 5 min NOTE: Use heated lid 5. When temperature reaches 4 C transfer plate to the bench and let incubate at RT 5 min. to bind RNA to beads. 6. Remove adhesive seal. 7. Transfer plate to magnetic stand for 5 min. at RT. 8. Remove and discard all of the supernatant. Do not disturb pellets. 9. Remove plate from magnetic stand 10. Wash beads by adding 200ul of Bead Washing Buffer (BWB) to each well and pipette up and down 6xs to resuspend beads. 11. Return the plate to the magnetic stand for 5 min at RT. 12. Briefly centrifuge the thawed Elution Buffer (ELB) to 600xg 5 sec. 13. Remove BWB from each well of plate and discard. Do Not Disturb Pellets. 14. Remove plate from magnetic stand. 15. Add 50ul of Elution Buffer (ELB) to each well of the plate. Pipette up and down 6xs to suspend beads. - Store stock Elution Buffer (ELB) at 4 C 16. Seal plate with adhesive seal. 17. Incubate the sealed plate in a thermal cycler using the following profile to elute mrna from Beads: ELUTE 80 C 2 min NOTE: Use Heated Lid 25 C Hold 18. Remove plate from the thermal cycler when 25 C is reached and place at RT. Remove adhesive seal. 19. Add 50ul of Bead Binding Buffer to each well of the plate to allow the RNA to re-bind to the beads. Pipette up and down 6xs to thoroughly mix beads. 20. Incubate at RT 5 min. -Store Bead Binding Buffer at 4 C 21. Place plate on magnetic stand at RT 5 min. 22. Remove and discard supernatant without disturbing beads. 23. Remove plate from magnetic stand and wash beads with 200ul Bead Washing Buffer (BWB) by pipetting up and down 6xs. -Store the Bead Washing Buffer (BWB) at 4 C 24. Place plate on magnetic stand at RT for 5 min. 25. Remove and discard the supernatant from each well of the plate. Do Not Disturb Beads. 26. Remove plate from magnetic stand.

2 27. Add 19.5ul Fragment, Prime, Finish Mix to each well and mix up and down 6xs with a pipette. 2 NOTE: Fragment, Prime, Finish Mix contains the Random Hexamers for RT priming and serves as the 1st Strand cdna Synthesis Buffer. : Store the Fragment, Prime, Finish Mix at -25 C. 28. Seal the plate with an adhesive seal. 29. Incubate the sealed plate in a thermal cycler using the following profile to elute, fragment and prime the RNA: E2FP 94 C 8 min NOTE: Use Heated lid 30. Remove the plate when 4 C is reached and centrifuge briefly in plate adaptor at 15 C. 31. Proceed immediately to First Strand Synthesis Synthesize First Strand cdna NOTE: Thaw one tube of First Strand Synthesis Act D Mix, spin briefly and place on ice. NOTE: Apply a CDP barcode label to a new 0.3ml PCR plate NOTE: Aliquot SuperScipt II Supplemented First Strand Master Mix into strip tubes (with 10% excess volume) seal and keep on ice. Residual mix can be returned to the master vial. 1. Transfer the RNA Bead Plate (RBP) to the magnetic stand and incubate at RT for 5 min. DO NOT REMOVE PLATE FROM MAGNETIC STAND 2. Remove the adhesive seal from the plate. 3. Transfer 17ul of the supernatant (fragmented and primed mrna) from each well of the RBP to the corresponding well of a new plate labeled with the cdna Plate (CDP) barcode. NOTE : Some liquid may remain in the RBP wells. Avoid disturbing pellets and transferring beads. 4. Add 50ul SuperScript II to the thawed tube of First Strand Synthesis Mix. Mix well by finger flicking DO NOT VORTEX Centrifuge briefly. Label tube with SS+ and date to indicate that SuperScript has been added. NOTE : The final First Strand Synthesis Act D Mix is a 1:9 ratio of SuperScript II to First Strand Synthesis Act D Mix : The unused SuperScript II supplemented First Strand Master Mix can be stored at -25 C and used for up to six subsequent mrna Library constructions. 5. Add 8ul of supplemented First Strand Synthesis Act D Mix to each well of the CDP plate. Gently mix by pipetting up and down 6xs. 6. Seal the plate with an adhesive seal and centrifuge briefly at 15 C. 7. Incubate the CDP plate in a thermal cycler using the following profile: FIRST 25 C 10 min NOTE : Use Heated Lid at 100 o C 42 C 15 min 70 C 15 min 8. When the cycler reaches 4 C remove CDP plate and proceed immediately to the Second Strand Synthesis.

3 Synthesize Second Strand cdna 3 NOTE: Prepare a fresh stock of 80% ETOH NOTE: Thaw at RT one tube each of Second Strand Marking Master Mix. Warm 4 C Resuspension Buffer to RT.. NOTE: Remove one tube of End Repair Control from -20 C and thaw at RT. : Use of the End Repair Control is optional and can be replaced with the same volume of resuspension buffer. NOTE: Warm AMPure Beads to RT 30 min. vortex until well dispersed. Review AMPure XP handling guidelines. NOTE: Initiate thermal cycler profile SSM16 and pre-heat to 16 C. 1. Briefly Centrifuge Thawed Second Strand Marking Master Mix. 2. Remove the adhesive seals from the CDP (cdna plate). 3. Dilute the End Repair Control 1/50 in Resuspension Buffer (2ul of End Repair Control + 98ul of Resuspension buffer). 4. Add 5ul of diluted End Repair Control (or 5ul Resuspension buffer if control is not used) into each well of the plate. Discard unused diluted End Repair Control. 5. Add 20ul of thawed Second Strand Marking Master Mix to each well of the CDP plate. Gently pipette up and down 6xs to mix thoroughly. NOTE: Aliquot Second strand marking master mix into a strip tube with 10% excess for aliquoting into CDP plate. Remaining reagent can be returned to stock tube and stored at -25 C. 6. Seal plate with an adhesive seal. 7. Incubate the CDP plate on a preheated thermal cycler with closed lid at 16 C 1 hour using the following profile : SSM16 16 C 1 hour NOTE: Do Not use heated lid 16 C hold 8. Remove the CDP plate and place at RT to equilibrate. Remove adhesive seal. 9. Vortex the RT AMPure XP beads until fully dispersed then add 90ul of beads to each well of the CDP plate containing 50ul of ds cdna. Pipette up and down gently 10xs to mix. 10. Incubate the CDP plate at RT 15 min. 11. Place the CDP plate on the magnetic stand at RT for 5 min make sure beads are completely deposited on side of well. 12. Remove and discard 135ul of the supernatant from each well. NOTE: Some liquid may remain in the wells DO NOT DISTURB BEADS 13. Leave CDP plate on the magnetic stand and wash wells with 200ul freshly prepared 80% ETOH DO NOT DISTURB BEADS 14. Incubate CDP plate at RT 30 sec. Remove ETOH using pipette - DO NOT DISTURB BEADS 15. Repeat 80% ETOH wash. 16. Let CDP plate stand at RT 15 min to dry then remove plate from magnetic stand. 17. Briefly centrifuge the RT Resuspension Buffer 18. Add 17.5ul Resuspension Buffer to each well of the CDP plate. Mix up and down 10xs to completely resuspend beads. 19. Incubate the CDP plate at RT for 2 min. 20. Place the CDP plate on the Magnetic Stand at RT for 5 min. 21. Transfer 15ul of the supernatant containing the ds cdna to a new 0.3ml plate labeled with an ALP Barcode. NOTE: Some liquid may remain in the wells DO NOT DISTURB BEADS Safe Stopping Point: Seal ALP plate with adhesive seal and store at -20 C for up to 7 days.

4 Adenylate 3 Ends 4 NOTE: Remove one tube of A-Tailing Mix and A-Tailing Control from -20 C and thaw at RT. : Use of the A-Tailing Control is optional and can be replaced with the same volume of resuspension buffer. : If the A-Tailing Mix and A-Tailing Control reagents are not consumed in one use, transfer remaining reagents into single use aliquots and label tubes. Store at -20 C. NOTE: Remove ALP plate from -20 C storage and thaw at RT. Briefly centrifuge ALP plate at 280xg for 1 min the remove adhesive seal. NOTE: Initiate thermal cycler profile ATA70 and preheat to 37 C. NOTE: Aliquot the appropriate volume of each reagent (with 10% excess per tube) into strip tubes. Cap tubes and keep on ice until needed. Remaining content can be re-store at -20 C. 1. Briefly mix and centrifuge the thawed A-Tailing Control tube. 2. Dilute the A-Tailing Control 1/100 in Resuspension Buffer (1ul A-Tailing Control + 99ul Resuspension Buffer) 3. Add 2.5ul of diluted A-Tailing Control (or 2.5ul Resuspension Buffer if control is not used) to each well of the ALP plate. Discard unused diluted A-Tailing Control. 4. Set pipette to 30ul fill volume and gently pipette up and down 10xs to mix. 5. Add 12.5ul of thawed A-Tailing Mix to each well of the ALP plate. Pipette up and down 10xs to mix. 6. Seal ALP plate with adhesive sea. 7. Incubate the ALP plate in the pre-heated thermal cycler with closed lid at 37 C for 30 min using the following profile:ata70 37 C 30 min NOTE: use heated lid at 100 o C 70 o C 5 min 8. Immediately remove the ALP plate from the thermal cycler Proceed Immediately to Ligate Adapters. Ligate Adapters NOTE: Remove the appropriate RNA Adapter Index tubes (AR001-AR012, depending on the RNA Adapter Indexes being used) and one tube of Stop Ligase Buffer and Ligase Control from -20 C and thaw at RT. : Use of the Ligase Control is optional and can be replaced with the same volume of resuspension buffer. : If the Ligase Control will not consumed in one use, transfer remaining reagents into single use aliquots and label tubes. Store at -20 C. NOTE: Remove Resuspension Buffer from 4 C and warm to RT. NOTE: Remove the AMPure XP Beads from 4 C storage and warm to RT at least 30 min. NOTE: Initiate thermal cycler profile ALP30 and pre-heat to 30 C. NOTE: Apply a CAP barcode label to a new 0.3ml PCR plate. NOTE: Apply a PCR barcode label to a new 0.3ml PCR plate. NOTE: Aliquot the appropriate volume of each reagent (with 10% excess per tube) into strip tubes. Cap tubes and keep on ice until needed. Remaining content can be re-store at -20 C. 1. Briefly centrifuge the thawed RNA Adapter Index tubes, Ligase Control and Stop Ligase Mix. 2. Remove the adhesive seal from the ALP plate. 3. Dilute the Ligase Control 1/100 in Resuspension Buffer (1ul Ligase Control +99ul Resuspension Buffer). 4. Add 2.5ul of diluted Ligase Control (or 2.5ul Resuspension Buffer if control is not used) to each well of the ALP plate. Discard the unused diluted Ligase Control. 5. Remove the DNA Ligase Mix from -20 C immediately before use and leave in -20 C benchtop storage cooler.

5 6. Add 2.5ul of DNA Ligase Mix directly from the -20 C benchtop storage cooler to each well of the ALP plate. Return -20 C benchtop storage cooler back to freezer immediately after use. 7. Add 2.5ul of the appropriate thawed RNA Adapter Index (AR001-AR012) to each well of the ALP plate. 8. Adjust the pipette to 40ul and gently pipette the entire volume up and down 10xs to mix thoroughly. 9. Seal the ALP plate with an adhesive seal. 5 NOTE: When indexing libraries, Illumina recommends arranging samples that will be combined into a common pool in the same row. Each column should contain a common index (ie AR007). This will facilitate pipeting operations when dispensing indexed adapters and pooling indexed libraries later. 10. Incubate the ALP plate in the pre-heated thermal cycler with closed lid at 30 C for 10 min using the following profile:alp30 30 C 10 min NOTE: use heated lid at 100 o C 30 C Hold 11. Remove the ALP plate from the thermal cycler. 12. Remove the adhesive seal from the ALP plate. 13. Add 5ul Stop Ligase Buffer to each well of the ALP plate and mix by gently pipetting up and down 10xs. 14. Vortex the pre-warmed AMPure XP Beads until fully dispersed. 15. Add 42ul of beads to each well of the ALP plate. Gently pipette up and down 10xs to mix well. 16. Incubate the ALP plate at RT 15 min. 17. Place the ALP plate on the magnetic stand at RT for at least 5 min making sure liquid clears. 18. Remove and discard 79.5ul of supernatant from each well. DO NOT DISTURB BEADS. Change tips after each removal. NOTE: Leave plate on the magnetic stand while performing the 80% ETOH washing steps. 19. Add 200ul freshly prepared 80% ETOH to each well without disturbing the beads. 20. Incubate the ALP plate at RT for at least 30 sec. then remove and discard all the supernatant from each well without disturbing the beads. Change tips between wells. 21. Repeat ETOH wash. 22. Incubate the ALP plate on the magnetic stand at RT 15 min to dry residual ETOH. 23. Remove plate from magnetic stand and resuspend the dry pellet in each well with 52.5ul of Resuspension Buffer. Gently pipette up and down 10xs to mix. 24. Incubate the ALP plate at RT for 2 min. 25. Place the ALP plate on the magnetic stand and incubate at least 5 min - make sure liquid clears. 26. Transfer 50ul of clear supernatant from each well of the ALP plate to the corresponding well of the new 0.3ml plate with the CAP barcode. Some residual liquid may remain in each well. 27. Vortex the AMPure XP beads to disperse and add 50ul of beads into each well of the CAP plate for a second clean-up. Pipette up and down 10xs to mix thoroughly. 28. Incubate the CAP plate at RT for 15 min. 29. Place the CAP plate on the magnetic stand at RT for 5 min making sure the liquid has cleared. 30. Remove and discard 95ul of the supernatant from each well without disturbing the beads. Some liquid may remain in each well. Remember to change tips between wells. NOTE: Leave plate on the magnetic stand while performing the 80% ETOH washing steps. 31. Add 200ul freshly prepared 80% ETOH to each well of the CAP plate without disturbing the beads. 32. Incubate the CAP plate at RT for at least 30 sec, then remove and discard all of the supernatant from each well without disturbing beads. Change tips after each removal. 33. Repeat ETOH wash. 34. Incubate the CAP plate on the magnetic stand at RT 15 min to dry residual ETOH. 35. Remove plate from magnetic stand and resuspend the dry pellet in each well with 22.5ul of Resuspension Buffer. Gently pipette up and down 10xs to mix. 36. Incubate the CAP plate at RT 2 min. 37. Place the CAP plate on the magnetic stand at RT for at least 5 min making sure liquid clears. 38. Transfer 20ul of clear supernatant from each well of the CAP plate to the corresponding well of the new 0.3ml PCR plate with the PCR barcode. Some residual liquid may remain in each well. Safe Stopping Point: Seal PCR plate with adhesive seal and store at -20 C for up to 7 days.

6 Enrich DNA Fragments 6 NOTE: Remove one tube each of PCR Master Mix and PCR Primer Cocktail from -20 C and thaw at RT - centrifuge briefly. NOTE: Remove the AMPure XP Beads from 4 C and warm to RT at least 30 min. NOTE: Remove Resuspension Buffer and the PCR plate from -20 C and thaw to RT. Briefly centrifuge plate at 280xg for 1 min. NOTE: Initiate thermal cycler profile PCR15 and pre-heat to 98 C. NOTE: Apply a TSP1 barcode to a new 0.3ml PCR plate NOTE: Aliquot the appropriate volume of each reagent (with 10% excess per tube) into strip tubes. Cap tubes and keep on ice until needed. Remaining content can be re-store at -20 C. 1. Add 5ul of thawed PCR Primer Cocktail to each well of the PCR plate. 2. Add 25ul of thawed PCR Master Mix to each well of the PCR plate. Pipette up and down 10xs to mix thoroughly. 3. Cover plate with adhesive seal. 4. Incubate the PCR plate in the pre-heated thermal cycler with closed lid using the following profile: PCR15 1 cycle 98 C 30 sec NOTE: use heated lid at 100 o C 15 cycles 98 C 10 sec 60 C 30 sec 72 C 30 sec 1 cycle 72 C 5 min 5. Vortex the pre-warmed AMPure Beads until they are completely dispersed. 6. Add 50ul of the beads to each well of the PCR plate containing 50ul of PCR amplified library and pipette up and down 10xs to mix thoroughly. 7. Incubate the PCR plate at RT 15 min. 8. Transfer the PCR plate to the magnetic stand at RT for at least 5 min making sure the liquid clears. 9. Remove and discard 95ul of the supernatant from each well without disturbing the beads. Some liquid may remain in the wells. Remember to change tips. NOTE: Leave plate on the magnetic stand while performing the 80% ETOH washing steps. 10. Add 200ul of freshly prepared 80% ETOH to each well without disturbing the beads. 11. Incubate the PCR plate at RT at least 30 sec then remove and discard all the supernatant from each well without disturbing the beads. Remember to change tips. 12. Repeat ETOH wash. 13. Incubate the PCR plate on the magnetic stand at RT 15 min to dry residual ETOH. 14. Remove plate from magnetic stand and resuspend the dry pellet in each well with 32.5ul of Resuspension Buffer. Gently pipette up and down 10xs to mix. 15. Incubate the PCR plate at RT for 2 min. 16. Place the PCR plate on the magnetic stand at RT for at least 5 min making sure liquid clears. 17. Transfer 30ul of clear supernatant from each well of the PCR plate to the corresponding 1.5 ml non-sticky Ambion microcentrifuge tube. Some residual liquid may remain in each well. Safe Stopping Point: Store amplified libraries at -20 C for up to 7 days.

7 Validate Library 7 1. Determine the concentration of each amplified library using the Nanodrop. 2. Perform QC of the amplified library by running 1ul of each sample on the Agilent 2100 Bioanalyzer using the Agilent DNA 1000 Chip. NOTE: The final product should be a band at approximately 280bp for a single read library 3. Calculate the nm concentration of each library. 4. Transfer 10ul of each library to a MIDI plate labeled with a DCT barcode and dilute to 4nM concentration using EB buffer (10mM Tris HCL ph 8.5) supplemented with 0.1% Tween Pipet up and down with pipette 10xs to mix. 6. For non-multiplexed samples that do not require pooling proceed to cluster generation. NOTE: Libraries diluted to 4nM in EB buffer supplemented 0.1% Tween20 are safe for long term storage at -20 C Pooling Libraries (Optional) 1. Apply a PDP barcode label to a new 0.3ml plate (for multiplexing only) 2. Determine the samples to be combined together for each pool. NOTE: Verify that libraries to be pooled have unique indexes!! 3. Transfer 10 ul of each normalized library to be pooled from the DCT plate to one well of the new PDP labeled plate. The total volume of the combines sample libraries will 10x the number of combined samples (6 libraries = 60ul). 4. Pipette the entire volume up and down 10x to thoroughly mix the pooled libraries. 5. Proceed to cluster generation