USC SCAP-T Protocols

USC NUGEN LIBRARY PREP AND CDNA SYNTHESIS PROTOCOL VERSION 1

The Collected RNA will be kept in -­80’C

cDNA synthesis(From Nugen RNAseq V2) & 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 
    (65ºC – 2 min, hold at 4ºC or 65ºC – 5 min, 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. 
    Per sample combine: 2.5 µL Buffer Mix A2 + 0.5 µL Enzyme Mix A3. Mix well.
  7. Add 3 µL of First Strand Master Mix to each tube, mix by pipetting, spin and place on ice.
  8. Place the tubes in a thermal cycler running Program Nu2 
    (4ºC – 1 min, 25ºC – 10 min, 42ºC – 10 min, 70ºC – 15 min, hold at 4ºC).
  9. Once the thermal cycler reaches 4ºC, remove tubes, spin and place on ice.
  10. 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).
  11. Spin B2 ver 2 briefly and place on ice. Vortex B1 ver 3, spin and place on ice.
  12. Prepare Second Strand Master Mix. Be sure to pipet B2 enzyme slowly. 
    Per sample combine: 9.7 µL Buffer Mix B1 + 0.3 µL Enzyme Mix B2. Mix well.
  13. Add 10 µL of Second Strand Master Mix to each reaction tube, mix by pipetting, spin and place on ice.
  14. Place the tubes in a thermal cycler running Program Nu3 (4ºC – 1 min, 25ºC – 10 min, 50ºC – 30 min, 80ºC – 20 min, hold at 4ºC).
  15. Once the thermal cycler reaches 4ºC, remove tubes, spin and place on bench top.
  16. At room temperature, add 32 µL of RNAClean XP beads to each reaction tube and mix by pipetting 10 times.
  17. Incubate at room temperature for 10 minutes.
  18. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  19. Remove only 45 µL of the binding buffer.
  20. Add 200 µL of freshly prepared 70% ethanol and let stand for 30 seconds. Remove the ethanol using a pipette.
  21. Repeat the ethanol wash 2 more times.
  22. Remove all excess ethanol after the final wash and let beads air dry for 15 to 20 minutes.
  23. Ensure the tubes have completely dried and no residual ethanol is left.
  24. 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.
  25. Prepare SPIA Master Mix. 
    Per sample combine: 20 µL Buffer Mix C2 + 10 µL Primer Mix C1 + 10 µL Enzyme Mix C3. Mix well.
  26. Add 40 µL of SPIA Master Mix to each reaction tube and resuspend beads thoroughly by pipetting. Place on ice.
  27. Place the tubes in a thermal cycler running Program Nu4 
    (4ºC – 1 min, 47ºC – 70 min(modified), 80ºC – 20 min, hold at 4ºC).
  28. At room temperature, add 72 µL of RNAClean XP beads to each reaction tube and mix by pipetting 10 times.
  29. Incubate at room temperature for 10 minutes.
  30. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  31. Remove only 110 µL of the binding buffer.
  32. Add 200 µL of freshly prepared 70% ethanol and let stand for 30 seconds. Remove the ethanol using a pipette.
  33. Repeat the ethanol wash 2 more times.
  34. Remove all excess ethanol after the final wash and let beads air dry for 15 to 20 minutes.
  35. Ensure the tubes have completely dried and no residual ethanol is left.
  36. Resuspend in 50ul of nuclease free water.
  37. Transfer the tubes to the magnet and let stand for an additional 5 minutes.
  38. Carefully transfer 48ul into a Covaris Tubes and 1ul into 8-­-well strip tubes.
  39. On 8-­-well strip tubes, add 3ul of D1000 buffer from Agilent 2200 Tapestation reagents and vertex vigorously.
  40. Place D1000 Tapes, strip tubes and pipet tips on Agilent 2200 Tapestation, remove caps and run it.
  41. The Covaris tubes will be processed with protocol for 325bp. 

 

Library Construction (Nugen Mondrian Rapid)

  1. Remove all liquid from Covaris tubes and take 1ul for tapestation.
  2. Dry down the 46ul of sample to 21.4ul with Speedvac.

Library Construction

  1. Take out the Agencourt RNAClean XP beads from 4’C
  2. Prepare sample solution for loading onto the cartridge (this is done on a per sample basis and not as a master mix) according to the volumes shown in Table. You must prepare and process no fewer than eight samples on each cartridge.

COMPONENT VOLUME

cDNA 21.4 µL 
Agencourt RNAClean XP beads 3.6 µL
Sample Concentration Solution 25 µL
Nuclease-­-free water (D1) to 50 µL final
  1. ​Thaw End Repair Buffer Mix (ER1 ver 6) at room temperature, vortex to mix well and spin down briefly. Keep End Repair Enzyme Mix (ER2 ver 4) on ice.
  2. Prepare master mix in a 0.5 mL microcentrifuge tube or 0.2 mL PCR tube according to the volumes shown in Table below.
Component Vial Cap Volume  
End Repair Buffer Mix (ER1 ver 6) 7.0 µL
End Repair Enzyme Mix (ER2 ver 4) 3.0 µL
Total volume 10.0 µL 
  1. Mix well by carefully pipetting, avoiding the introduction of bubbles. Briefly spin down to bring the master mix to the bottom of the tube.
  2. Thaw End Repair Enhancer Buffer Mix (ER4) at room temperature, vortex to mix well and spin down briefly. Keep End Repair Enhancer (ER3) on ice.
  3. Prepare master mix in a 0.5 mL microcentrifuge tube or 0.2 mL PCR tube according to the volumes shown in Table below. Mix well by carefully pipetting, avoiding the introduction of bubbles. Briefly spin down to bring the master mix to the bottom of the tube.
Component Vial Cap Volume  
End Repair Buffer Mix (ER4) 7.0 µL
End Repair Enzyme Mix (ER3) 3.0 µL
Total volume 10.0 µL
  1. Thaw Ligation Buffer Mix (L1 ver 5) at room temperature, vortex to mix well and spin down briefly. Keep Ligation Enzyme Mix (L3 ver 4) on ice.
  2. Prepare master mix in a 0.5 mL microcentrifuge tube or 0.2 mL PCR tube according to the volumes shown in Table below. Mix well by carefully pipetting, avoiding the introduction of bubbles. Briefly spin down to bring the master mix to the bottom of the tube. 
Component Vial Cap Volume  
Ligation Buffer Mix (L1 ver 5) 7.0 µL
Ligation Enzyme Mix (L3 ver 4) 3.0 µL
Total volume 10.0 µL
  1. Thaw Final Repair Buffer Mix (FR1 ver 2) at room temperature, vortex to mix well and spin down briefly. Keep Final Repair Enzyme Mix (FR2 ver 2) on ice.
  2. Prepare master mix in a 0.5 mL microcentrifuge tube or 0.2 mL PCR tube according to the volumes shown in Table below. Mix well by carefully pipetting, avoiding the introduction of bubbles. Briefly spin down to bring the master mix to the bottom of the tube.
Component Vial Cap Volume  
Final Repair Buffer Mix (FR1 ver 2) 8.0 µL
Final Repair Enzyme Mix (FR2 ver 2) 2.0 µL
Total volume 10.0 µL
  1. Thaw ligation adaptors at room temperature, vortex well to mix and spin downbriefly. 
  • For non-­-barcoded (non-­-multiplexed) samples, use vial L2 ver 10 (yellow vial cap).
  • For barcoded (multiplexed) samples, use the desired barcode adaptors(L2V9DR-­-BC1–16, yellow vial cap). 

The adaptors will be loaded onto the cartridge as indicated below (Step 8 of the Mondrian SP Cartridge Loading Instructions). 

  1. Load Adaptors, Mixes and samples to the Mondrian Cartridge and load the program.
  2. Collect droplets with library after the run.
 

 

© 2015 University of Pennsylvania