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Australia Mytilus EecSeq Probe Synthesis Day 4

Third Day of Stranded mRNA Seq Library Prep

All steps in are done side by side with Amy Zyck, continuing with sample tubes AL, FR, and MN, and Amy with TZ and RI

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

Library Amplification and Cleanup

  1. Removed sample tubes from 4 degree and get out 20μM indexes
  2. Added 12.5μl of KAPA HiFi Hot-Start Ready Mix to each tube
  3. Added 1.25μl each of the appropriate Index 1 and Index 2 to each tube:
    • AL - 501, 701
    • FR - 502, 702
    • MN - 503, 703
    • TZ - 504, 704
    • RI - 505, 705
  4. Pipetted to mix
  5. Put in thermocycler PCR program
  6. Took out KAPA Pure Beads and made fresh 80% EtOH
    At Post-PCR Bench
  7. Added 25μl KAPA Pure Beads to each sample tube and pipetted to mix
  8. Incubated for 15 minutes on shaker
  9. Placed tubes on magnet plate
  10. Removed 45μl of supernatant from each tube
  11. Added 100μl 80% EtOH to each tube
  12. Removed 100μl of supernatant from each tube
  13. Added 100μl 80% EtOH to each tube
  14. Removed ALL of the supernatant from each tube, using a p20 pipette tip to get rid of droplets
  15. Resuspended beads in 22μl 10mM Tris HCl pH 8
  16. Placed tubes on shaker for 5 minutes
  17. Placed tubes back on magnet plate and removed 10μl of clear supernatant into new tubes

Qubit and TapeStation of finished Libraries

Broad Range Qubit
199μl BR buffer * 7.5 = 1492.5μl
1μl BR reagent * 7.5 = 7.5μl

S1 S2 AL FR MN TZ RI
171.16 RFU 18808 RFU 25.2 ng/μl 21.5 ng/μl 28.2 ng/μl 35.8 ng/μl 11.9 ng/μl
    25.4 ng/μl 21.6 ng/μl 29 ng/μl 36.6 ng/μl 12.2 ng/μl

D5000 TapeStation

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Written on June 13, 2019