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Australia Mytilus EecSeq Probe Synthesis Day 2

First day of Stranded mRNA Seq Library Prep

All steps in are done side by side with Amy Zyck, where I would do sample tubes AL, FR, and MN, and she would do TZ and RI

Following half reactions from the KAPA Biosystems Stranded mRNA-Seq Kit, also see the EeqSeq protocol

  1. Took out mRNA capture beads, mRNA bead binding buffer, and mRNA bead wash buffer from 4 degree and let get to room temp
  2. Resuspended mRNA capture beads by pipetting up and down
  3. Calculated the number of beads needed:
    26.25μl beads * 5 samples = 131.25μl
  4. Pipetted 131.25μl mRNA capture beads into a new 1.5mL tube and put on the large magnet stand
  5. Removed 127μl of supernatant
  6. Added 131.25μl of bead binding buffer and pipetted to mix off magnet
  7. Put back on magnet and removed as much supernatant as possible
  8. Added 131.25μl of bead binding buffer again and pipetted to mix off magnet
  9. Put back on magnet and again removed as much supernatant as possible
  10. Added 131.25μl of bead binding buffer and pipetted to mix off magnet
  11. Added 25μl of the resuspended mRNA capture beads to each sample tube (each locality pool is 25μl in a PCR strip tube)
  12. Placed tubes in the 1st mRNA capture program in the thermocycler
  13. Placed tubes on the magnet plate and removed all supernatant when the solution went clear
  14. Removed tubes from the magnet plate and resuspended beads in 100μl of bead wash buffer
  15. Placed tubes on the magnet plate and removed all of the supernatant when the solution went clear
  16. Resuspended beads in 25μl of RNase-free water off magnet
  17. Placed tubes in the 2nd mRNA capture program in the thermocycler
  18. Took tubes out of the thermocycler and added 25μl of bead binding buffer to each tube and pipetted to mix
  19. Incubated the tubes are 20C for 5 minutes (program in thermocycler for this)
  20. Made 1X Fragment, Prime, and Elute Buffer on ice bucket:
    5.5μl water * 5.2 = 28.6μl
    5.5μl 2X FPE buffer * 5.2 = 28.6μl
  21. Placed tubes on the magnet plate and removed supernatant when the solution went clear
  22. Resuspended beads off magnet in 11μl 1X FPE buffer
  23. Put in thermocycler for RNA fragmentation program (7 minutes ate 94C)
  24. Made 1st Strand Synthesis Master Mix on ice:
    5.5μl 1st strand synthesis buffer * 5.2 = 28.6μl
    .5μl KAPA script * 5.2 = 2.6μl
  25. IMMEDIATELY placed tubes on magnet plate once the program was finished (some tubes had popped open)
  26. Removed 10μl of clear supernatant and placed in new PCR strip tubes on ice
  27. Added 5μl of the 1st strand synthesis master mix to each tube and pipetted to mix
  28. Placed in thermocycler 1st strand synthesis program
  29. Made 2nd Strand Synthesis and Marking Master Mix on ice:
    15.5μl 2nd strand marking buffer * 5.2 = 80.6μl
    1μl second strand enzyme * 5.2 = 5.2μl
  30. Removed tubes from the thermocycler and placed on ice
  31. Added 15μl of the 2nd strand synthesis and marking master mix and pipetted to mix
  32. Put in thermocycler 2nd strand synthesis program
  33. Took KAPA Pure Beads out of the 4 degree and swirled them to mix and let get to room temp
  34. Took tubes out of the thermocycler and added 54μl KAPA Pure Beads, pipetting to mix
  35. Incubated tubes on shaker for 15 minutes
  36. Made A-tailing Safe Stopping Point Master Mix on ice:
    6.75μl water * 5.2 = 35.1μl
    0.75μl 10X A-tailing buffer * 5.2 = 3.9μl
  37. Made fresh 80% EtOH for that day
  38. Placed tubes on magnet plate and removed 80μl clear supernatant
  39. Added 200μl 80% EtOH to each tube
  40. Removed EtOH from each tube
  41. Added 200μl 80% EtOH to each tube
  42. Removed ALL EtOH from each tube, using a p20 to get extras and droplets of EtOH
  43. Waited ~30 seconds or less and resuspended the beads in 7.5μl of the A-tailing Safe Stopping Point Master Mix
  44. Spun tubes down to make sure all the beads were off the sides
  45. Placed tubes in 4 degree overnight
Written on June 11, 2019