USC SCAP-T Protocols

USC LIBRARY PREP AND CDNA SYNTHESIS PROTOCOL VERSION 2

NuGEN Protocol

Part I . cDNA synthesis(From Nugen RNAseq V2) and Covaris Shearing

Part II. Purification and removal of 135 peak

Part III. Library Construction (Nugen Ovation Rapid)

 

 

Part I . cDNA synthesis(From Nugen RNAseq V2) and Covaris Shearing

  1. Spin A3 ver 1 briefly and place on ice. Vortex A1 ver 4 and A2 ver 3, spin and place on ice from Nugen RNAseq V2 kit.
  2. On ice, mix 2 µL of A1 and 5 µL of total RNA sample in a 0.2 mL PCR tube.
  3. Place the tubes in a thermal cycler running Program Nu1 a. 65ºC – 2 min, hold at 4ºC or 65ºC – 5 min b. hold at 4ºC
  4. Once the thermal cycler reaches 4°C, remove tubes and place on ice.
  5. Prepare First Strand Master Mix (calculated volumes allow for appropriate overfill).
  6. Be sure to pipet A3 enzyme slowly and rinse out tip at least five times into buffer.

 

Buffer Mix (A2)  2.5 µL
Enzyme Mix (A3)  0.5 µL
Total volume 5.0 µL
  1. Add 3 µL of First Strand Master Mix to each tube, mix by pipetting, spin and place on ice.
  2. Place the tubes in a thermal cycler running Program Nu2
    1. 4ºC – 1 min
    2. 25ºC – 10 min
    3. 42ºC – 10 min
    4. 70ºC – 15 min
    5. hold at 4ºC
  3. Once the thermal cycler reaches 4ºC, remove tubes, spin and place on ice.
  4. Resuspend the Agencourt® RNAClean® XP beads provided with the Ovation RNASeq System V2 and leave at room temperature for use in the next step. Also, thaw the Second Strand cDNA Synthesis reagents (yellow).
  5. Spin B2 ver 2 briefly and place on ice. Vortex B1 ver 3, spin and place on ice.
  6. Prepare Second Strand Master Mix. Be sure to pipet B2 enzyme slowly.

 

Buffer Mix (B1)  9.7 µL
Enzyme Mix (B2) 0.3 µL
Total volume 10.0 µL
  1. Add 10 µL of Second Strand Master Mix to each reaction tube, mix by pipetting, spin and place on ice.
  2. Place the tubes in a thermal cycler running Program Nu3
    1. 4ºC – 1 min
    2. 25ºC – 10 min
    3. 50ºC – 30 min
    4. 80ºC – 20 min
    5. hold at 4ºC
  3. Once the thermal cycler reaches 4ºC, remove tubes, spin and place on bench top.
  4. At room temperature, add 32 µL of RNAClean XP beads to each reaction tube and mix by pipetting 10 times.
  5. Incubate at room temperature for 10 minutes.
  6. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  7. Remove only 45 µL of the binding buffer.
  8. Add 200 µL of freshly prepared 70% ethanol and let stand for 30 seconds. Remove the ethanol using a pipette.
  9. Repeat the ethanol wash 2 more times.
  10. Remove all excess ethanol after the final wash and let beads air dry for 15 to 20 minutes.
  11. Ensure the tubes have completely dried and no residual ethanol is left.
  12. Thaw the SPIA Amplification reagents (red). Invert C3 ver 7 5 times to mix, spin and place on ice. Vortex C1 ver 9and C2 ver 11, spin and place on ice.
  13. Prepare SPIA Master Mix. Mix well.

 

Buffer Mix (C2)  20 µL
Primer Mix (C1) 10 µL
Enzyme Mix (C3) 10 µL
Total volume 40 µL
  1. Add 40 µL of SPIA Master Mix to each reaction tube and resuspend beads thoroughly by pipetting. Place on ice.
  2. Place the tubes in a thermal cycler running Program Nu4
    1. 4ºC – 1 min
    2. 47ºC – 70 min(modified)
    3. 80ºC – 20 min 
    4. hold at 4ºC
  3. At room temperature, add 72 µL of RNAClean XP beads to each reaction tube and mix by pipetting 10 times.
  4. Incubate at room temperature for 10 minutes.
  5. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  6. Remove only 110 µL of the binding buffer.
  7. Add 200 µL of freshly prepared 70% ethanol and let stand for 30 seconds. Remove the ethanol using a pipette.
  8. Repeat the ethanol wash 2 more times.
  9. Remove all excess ethanol after the final wash and let beads air dry for 15 to 20 minutes.
  10. Ensure the tubes have completely dried and no residual ethanol is left.
  11. Resuspend in 50ul of nuclease free water.
  12. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  13. Transfer liquid (50ul) to brand new tube.
  14. On 8-­-well strip tubes, add 3ul of D1000 buffer from Agilent 2200 Tapestation reagents and vertex vigorously.
  15. Place D1000 Tapes, strip tubes and pipet tips on Agilent 2200 Tapestation, remove caps and run it.
  16. Carefully transfer 48ul into a Covaris Tubes and 1ul into 8-­-well strip tubes.
  17. The Covaris tubes will be processed with protocol for 325bp.

Part II. Purification and removal of 135 peak

  1. If there is 135 peak present, add 0.8X volume of beads to each sample(40ul for 50ul sample).
  2. Incubate at room temperature for 10 minutes.
  3. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  4. Remove 90 µL of the binding buffer.
  5. Add 200 µL of freshly prepared 70% ethanol and let stand for 30 seconds. Remove the ethanol using a pipette.
  6. Repeat the ethanol wash 2 more times.
  7. Remove all excess ethanol after the final wash and let beads air dry for 15 to 20 minutes.
  8. Ensure the tubes have completely dried and no residual ethanol is left.
  9. Resuspend in 50ul of nuclease free water.
  10. Rerun Agilent 2200 tapestation to check for bands.

Part III. Library Construction (Nugen Ovation Rapid)

  1. Adjust input sample volume to 10 µL with 1X TE (low EDTA) in a PCR tube or plate. Depending on how much of cDNA we have choose one of three
    1. If the concentration of cDNA < 3ng/ul, dry down samples to 10 µL
    2. If the concentration of cDNA > 3ng/ul and <10ng/ul, take 10 µL of sample and keep the rest
    3. If the concentration of cDNA > 50ng/ul, use amount so that the final amount of cDNA is 400ng. Add water to 10 µL
  2. Thaw ER1 ver 4 at room temperature. Mix by vortexing, spin and place on ice. Spin down contents of ER2 ver 4 and ER3, place on ice
  3. Prepare End Repair Mastermix

 

End Repair Buffer Mix (ER1 ver 3)  3.5 µL
End Repair Enzyme Mix (ER2 ver 4) 0.5 µL
End Repair Enhancer (ER3) 1.0 µL
Total volume 5.0 µL
  1. Add 5 µL of End Repair Mastermix and run NuER
    1. 25ºC – 30 min
    2. 70ºC – 10 min
    3. hold at 4ºC
  2. Remove the Ligation Reagents (yellow) and Nuclease-free Water D1 (green) from -20ºC storage.
  3. Thaw L1 ver 4, L2DR­-BC1 – L2DR­-BC16, and Nuclease­-free Water (D1) at room temperature. Mix by vortexing, spin and place on ice.
  4. Spin down L3 ver 4 and place on ice.
  5. Add 3 µL of the appropriate L2 Ligation Adaptor Mix to each sample. Mix thoroughly by pipetting
  6. Prepare Ligation Mastermix

 

Ligation Buffer Mix (L1 ver 4)  6.0 µL
Ligation Enzyme Mix (L3 ver 4) 1.5 µL
Nuclease Free Water (D1) 4.5 µL
Total volume 12.0 µL
  1. Add 12µL of ligation mastermix and run NuER/L
    1. 25ºC – 30 min
    2. 70ºC – 10 min
    3. hold at 4ºC
  2. Remove Final Repair reagents (purple) from -­20°C storage. Thaw FR1 at room temperature. Mix by vortexing and place on ice. Spin down FR2 and place on ice
  3. Prepare Final Repair Mastermix Final Repair Buffer Mix (FR1) 19.0 µL Final Repair Enzyme Mix (FR2 ) 1.0 µL Total volume 20.0 µL
  4. Add 20µL of Final Repair Mastermix and run NuF
    1. 72ºC – 2 min
    2. hold at 25ºC
  5. Ensure Agencourt RNAClean XP beads have completely reached room temperature.
  6. Resuspend beads by inverting and tapping tube. After resuspending, do not spin beads.
  7. Prepare fresh 70% ethanol wash solution.
  8. At room temperature, add 50 µL (1.0 volume) bead suspension to each reaction.
  9. Mix thoroughly by pipetting.
  10. Incubate at room temperature for 10 minutes.
  11. Transfer tubes to magnet and let stand for 5 minutes to completely clear solution of beads.
  12. Carefully remove 80 µL binding buffer and discard it.
  13. With tubes still on magnet, add 200 µL of freshly prepared 70% ethanol and allow to stand for 30 seconds.
  14. Remove 70% ethanol wash using a pipette.
  15. Repeat 70% ethanol wash.
  16. Air dry beads on magnet for 10 minutes. Inspect each tube carefully to ensure that all ethanol has evaporated.
  17. Remove tubes from magnet.
  18. Add 11 µL 1X TE (low EDTA) to dried beads. Mix thoroughly to ensure all beads are resuspended and let stand on bench top for 5 minutes.
  19. Transfer tubes to magnet and let stand for 5 minutes for beads to clear solution.
  20. Carefully remove 9 µL eluted material, ensuring as few beads as possible are carried over, transfer to a fresh set of tubes and place on ice.
  21. Use qPCR to quantify for sequencing 
 

 

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