browse by date or tag

Block 10 Stranded mRNA Library Prep

Stranded mRNA Library Prep for CASE EecSeq Block 10

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

Capture and Fragmentation

  1. Calculated number of mRNA capture beads needed for 12 samples
    • 26.25*12= 315μl beads
  2. Pipetted 315μl mRNA capture beads into a new 1.5mL tube and put on the large magnet stand
  3. Removed supernatant
  4. Added 315μl of bead binding buffer and pipetted to mix off magnet
  5. Put back on magnet and removed as much supernatant as possible
  6. Added 315μl of bead binding buffer again and pipetted to mix off magnet
  7. Put back on magnet and again removed as much supernatant as possible
  8. Added 315μl of bead binding buffer and pipetted to mix off magnet
  9. Prepared RNA samples: 1μg in 25μl with the rest made up with nuclease free water
Sample RNA qubit volume for 1000ng water to 25ul
B10T24J1 71 14.1 10.9
B10T24J2 50 20.0 5.0
B10T24J3 81.5 12.3 12.7
B10T24J4 52 19.2 5.8
B10T24J5 100 10.0 15.0
B10T24J6 80.5 12.4 12.6
B10T24J7 42.2 23.7 1.3
B10T24J8 49.5 20.2 4.8
B10T24J9 99.8 10.0 15.0
B10T24J10 88.5 11.3 13.7
B10T24J11 98 10.2 14.8
B10T24J12 48.8 20.5 4.5
  1. Added 25μl of the resuspended mRNA capture beads to each sample tube
  2. Placed tubes in the 1st mRNA capture program in the thermocycler
  3. Placed tubes on the magnet plate and removed all supernatant when the solution went clear
  4. Removed tubes from the magnet plate and resuspended beads in 100μl of bead wash buffer
  5. Placed tubes on the magnet plate and removed all of the supernatant when the solution went clear
  6. Resuspended beads in 25μl of RNase-free water off magnet
  7. Placed tubes in the 2nd mRNA capture program in the thermocycler
  8. Took tubes out of the thermocycler and added 25μl of bead binding buffer to each tube and pipetted to mix
  9. Incubated the tubes are 20C for 5 minutes (program in thermocycler for this)
  10. Made 1X Fragment, Prime, and Elute Buffer on ice bucket:
    • 5.5μl nuclease free water * 12.5 = 67.1μl
    • 5.5μl 2X FPE buffer * 12.5 = 67.1μl
  11. Placed tubes on the magnet plate and removed supernatant when the solution went clear
  12. Resuspended beads off magnet in 11μl 1X FPE buffer
  13. Put in thermocycler for RNA fragmentation program (7 minutes ate 94C)
  14. Note: at this step, a master mix for the 1st strand synthesis was supposed to have been made, however I used 2nd strand reagents on accident. 5μl of 2nd strand reagents were added to samples J01-J08. These samples were frozen at -80 while samples J09-J12 were carried through 1st and second strand synthesis. See J01-J08 progress below
    First and Second Strand Synthesis
  15. Made correct 1st strand synthesis master mix for 4 samples
    • 5.5μl 1st strand synthesis buffer * 4.2 = 23.1μl
    • .5μl KAPA script * 4.2 = 2.1μl
  16. IMMEDIATELY placed tubes on magnet plate once the program was finished
  17. Removed 10μl of clear supernatant and placed in new PCR strip tubes on ice
  18. Added 5μl of the 1st strand synthesis master mix to each tube and pipetted to mix
  19. Placed in thermocycler 1st strand synthesis program
  20. Made 2nd Strand Synthesis and Marking Master Mix on ice:
    • 15.5μl 2nd strand marking buffer * 4.2 = 65.1μl
    • 1μl second strand enzyme * 4.2 = 4.2μl
  21. Removed tubes from the thermocycler and placed on ice
  22. Added 15μl of the 2nd strand synthesis and marking master mix and pipetted to mix
  23. Put in thermocycler 2nd strand synthesis program
  24. Took KAPA Pure Beads out of the 4 degree
  25. Took tubes out of the thermocycler and added 54μl KAPA Pure Beads, pipetting to mix
  26. Incubated tubes on shaker for 15 minutes at room temp
  27. Made A-tailing Safe Stopping Point Master Mix on ice:
    • 6.75μl water * 4.2 = 28.35μl
    • 0.75μl 10X A-tailing buffer * 4.2 = 3.15μl
  28. Performed normal bead clean up with fresh 80% EtOH
  29. Resuspended the beads in 7.5μl of the A-tailing Safe Stopping Point Master Mix
  30. Spun tubes down to make sure all the beads were off the sides
  31. Placed tubes in 4 degree overnight

Salvaging Samples J01-J08
Recommendations from KAPA technical service: 3X cleanup, elute in 1X FPE buffer, 1 minute incubation at 65C, then proceed with 1st strand synthesis

  1. 3X cleanup, 15μl in each tube, so 45μl KAPA pure beads added to each sample
  2. Performed normal bead cleanup extra carefully
  3. Made 1X FPE buffer:
    • 5.5μl nuclease free water * 8.5 = 67.1μl
    • 5.5μl 2X FPE buffer * 8.5 = 67.1μl
  4. Elute and Resuspend beads in 11μl 1X FPE buffer and place on magnet
  5. Save 10μl of supernatant
  6. Placed tubes in the thermocycler at 65C for 1 minute
  7. Placed tubes on ice
  8. Made 1st strand synthesis master mix
    • 5.5μl 1st strand synthesis buffer * 8.5 = 46.75μl
    • .5μl KAPA script * 8.5 = 2.75μl
  9. Added 5μl of the 1st strand synthesis master mix to each tube and pipetted to mix
  10. Placed in thermocycler 1st strand synthesis program
  11. Made 2nd Strand Synthesis and Marking Master Mix on ice:
    • 15.5μl 2nd strand marking buffer * 8.5 = 131.75μl
    • 1μl second strand enzyme * 8.5 = 8.5μl
  12. Removed tubes from the thermocycler and placed on ice
  13. Added 15μl of the 2nd strand synthesis and marking master mix and pipetted to mix
  14. Put in thermocycler 2nd strand synthesis program
  15. Took KAPA Pure Beads out of the 4 degree
  16. Took tubes out of the thermocycler and added 54μl KAPA Pure Beads, pipetting to mix
  17. Incubated tubes on shaker for 15 minutes at room temp
  18. Made A-tailing Safe Stopping Point Master Mix on ice:
    • 6.75μl water * 8.5 = 56.03μl
    • 0.75μl 10X A-tailing buffer * 8.5 = 6.225μl
  19. Performed normal bead clean up with fresh 80% EtOH
  20. Resuspended the beads in 7.5μl of the A-tailing Safe Stopping Point Master Mix
  21. Placed in 4 degree until later that day

A-tailing and Adapter Ligation
All 12 samples now going forward

  1. Made A-tailing after safe stopping point master mix
    • 5.25μl nuclease-free water * 12.5 = 65.625μl
    • 0.75μl A-tailing buffer * 12.5 = 9.375μl
    • 1.5μl A-tailing enzyme * 12.5 = 18.75μl
  2. Took sample tubes out of the 4 degree and added 7.5μl of the A-tailing after safe stopping point master mix to each tube and pipetted to mix
  3. Put tubes in the thermocycler A-tailing program
  4. Made the Adapter Ligation Master Mix:
    • 8μl nuclease-free water * 12.5 = 100μl
    • 7μl ligation buffer * 12.5 = 87.5μl
    • 2.5μl DNA ligase * 12.5 = 31.25μl
  5. Added Ligation master mix and planned adapter to each sample, pipetted to mix:
Sample μl of LMM μl of Adapter (700μM)
B10T24J1 17.5 2.5 SAII_NO_N
B10T24J2 17.5 2.5 SAII_NO_N
B10T24J3 17.5 2.5 SAII_NO_N
B10T24J4 17.5 2.5 SAIIv2
B10T24J5 17.5 2.5 SAII_NO_N
B10T24J6 17.5 2.5 SAIIv2
B10T24J7 17.5 2.5 SAIIv2
B10T24J8 17.5 2.5 SAIIv2
B10T24J9 17.5 2.5 SAIIv2
B10T24J10 17.5 2.5 SAIIv2
B10T24J11 17.5 2.5 SAII_NO_N
B10T24J12 17.5 2.5 SAII_NO_N
  1. Placed tubes on shaker for 30 minutes at room temp
  2. Added 35μl of room temperature PEG to each sample and pipetted to mix
  3. Performed normal bead clean up with fresh 80% EtOH
  4. Resuspended beads in 25μl 10mM Tris HCl pH8
  5. Added 25μl of room temperature PEG to each tube and pipetted to mix
  6. Performed normal bead cleanup with fresh 80% EtOH
  7. Resuspended and eluted beads in 11μl 10mM Tris HCl pH8 and placed on magnet plate
  8. Removed 10μl of supernatant into new PCR tubes and placed in the freezer at -20 for the weekend

Library Amplification and Index Addition

  1. Set up amplification with KAPA Hot start ready mix and individual index pairs, added to the 10μl of sample. Reactions set up on ice
Sample μl of HSRM μl of Index 1 μl of Index 2
B10T24J1 12.5 1.25 501 1.25 701
B10T24J2 12.5 1.25 502 1.25 702
B10T24J3 12.5 1.25 503 1.25 703
B10T24J4 12.5 1.25 501 1.25 701
B10T24J5 12.5 1.25 504 1.25 704
B10T24J6 12.5 1.25 502 1.25 702
B10T24J7 12.5 1.25 503 1.25 703
B10T24J8 12.5 1.25 504 1.25 704
B10T24J9 12.5 1.25 505 1.25 705
B10T24J10 12.5 1.25 506 1.25 706
B10T24J11 12.5 1.25 505 1.25 705
B10T24J12 12.5 1.25 506 1.25 706
  1. Vortexed and spun down
  2. Samples J01-J08 were placed in the thermocycler PCR program for 14 cycles to compensate for inevitable loss, samples J09-J12 were placed in the thermocycler with the usual 12 cycle program
  3. After the PCR, 25μl of KAPA Pure beads (1X) was added to each sample and pipetted to mix
  4. Performed normal bead clean up with fresh 80% EtOH
  5. Resuspended and eluted beads in 22μl 10mM Tris HCl pH 8

QC

High Sensitivity Qubit

Sample Standard 1 Standard 2 Average DNA(ng/μl)
B10T24J1 46 26006 32.2
B10T24J2 - - 31.8
B10T24J3 - - 46.2
B10T24J4 - - 33.9
B10T24J5 - - 22.6
B10T24J6 - - 31.4
B10T24J7 - - 29.2
B10T24J8 - - 22.8
B10T24J9 - - 80.4
B10T24J10 - - 86.8
B10T24J11 - - 62.8
B10T24J12 - - 60.4

D5000 TapeStation

See full report here

1

Representative sample trace: 2

Written on September 16, 2019