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Block 10 DNA Library Prep

DNA Library Prep for CASE EecSeq Block 10

Using the KAPA HyperPrep DNA Library Prep Kit on 18 DNA samples in 10mM Tris HCl pH 8 and 500ng from block 10 of the CASE experiment. All samples were sonicated to ~150 basepairs by using the QSonica protocol with a 27 minute time setting, 15 seconds on 15 seconds off, and an amplitude of 25%. Samples were spun down every 6 minutes.

End Repair and A-tailing

  • Prepared end repair and a-tailing master mix:
    • ERAT buffer 3.5μl * 19 = 66.5μl
    • ERAT enzyme 1.5μl * 19 = 28.5μl
  • Made 18 PCR strip tubes each with 25μl of 500ng sheared DNA
  • Added 5μl of ERAT master mix to each sample
  • Vortexed and spun down
  • Placed samples in thermocyler A-tailing program in JONP login (~ 1 hour)

Adapter Ligation

  • Prepared ligation master mix:
    • ligation buffer 15μl * 19.5 = 292.5μl
    • DNA ligase 5μl * 19.5 = 97.5μl
    • nuclease free water 2.5μl * 19.5 = 48.7μl
  • Added ligation master mix and appropriate planned adapters to each sample. Adapters were added last to minimize adapter-adapter ligation
Sample LMM Adapter
S1 22.5μl 2.5μl 1
S2 22.5μl 2.5μl 2
S3 22.5μl 2.5μl 3
S4 22.5μl 2.5μl 4
J1 22.5μl 2.5μl 5
J2 22.5μl 2.5μl 6
J3 22.5μl 2.5μl 7
J4 22.5μl 2.5μl 8
J5 22.5μl 2.5μl 9
J6 22.5μl 2.5μl 10
J7 22.5μl 2.5μl 11
J8 22.5μl 2.5μl 12
J9 22.5μl 2.5μl 1
J10 22.5μl 2.5μl 2
J11 22.5μl 2.5μl 3
J12 22.5μl 2.5μl 4
J13 22.5μl 2.5μl 5
J17 22.5μl 2.5μl 6
  • Pipetted all samples up and down with the multichannel and spun down
  • Incubated on the shaker at room temp for 1 hour

0.8X Cleanup

  • Made fresh 80% EtOH
  • Took KAPA Pure Beads out of fridge beforehand to warm to room temp
  • After incubation at RT, added 44μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
  • Placed tubes on shaker at room temp for 15 minutes
  • Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed supernatant from each tube on the magnet plate without disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
  • Resuspended beads in 12.5μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed supernatant when clear to new labeled PCR strip tubes

Library Amplification

  • Every 3 samples get a different index primer pair for amplification, so I made 6 different master mixes
  • Amp MM A:
    • 12.5μl HotStart Ready mix * 3.2 = 40μl
    • 1.25μl 507 primer * 3.2 = 4μl
    • 1.25μl 707 primer * 3.2 = 4μl
  • Amp MM B:
    • 12.5μl HotStart Ready mix * 3.2 = 40μl
    • 1.25μl 508 primer * 3.2 = 4μl
    • 1.25μl 708 primer * 3.2 = 4μl
  • Amp MM C:
    • 12.5μl HotStart Ready mix * 3.2 = 40μl
    • 1.25μl 509 primer * 3.2 = 4μl
    • 1.25μl 709 primer * 3.2 = 4μl
  • Amp MM D:
    • 12.5μl HotStart Ready mix * 3.2 = 40μl
    • 1.25μl 510 primer * 3.2 = 4μl
    • 1.25μl 710 primer * 3.2 = 4μl
  • Amp MM E:
    • 12.5μl HotStart Ready mix * 3.2 = 40μl
    • 1.25μl 511 primer * 3.2 = 4μl
    • 1.25μl 711 primer * 3.2 = 4μl
  • Amp MM F:
    • 12.5μl HotStart Ready mix * 3.2 = 40μl
    • 1.25μl 512 primer * 3.2 = 4μl
    • 1.25μl 712 primer * 3.2 = 4μl
  • Prepared new PCR tubes for the amplification with the following:
Sample volume adapter added DNA of sample volume of Amp MM
S1 10μl 15μl Amp MM A
S2 10μl 15μl Amp MM A
S3 10μl 15μl Amp MM A
S4 10μl 15μl Amp MM B
J1 10μl 15μl Amp MM B
J2 10μl 15μl Amp MM B
J3 10μl 15μl Amp MM C
J4 10μl 15μl Amp MM C
J5 10μl 15μl Amp MM C
J6 10μl 15μl Amp MM D
J7 10μl 15μl Amp MM D
J8 10μl 15μl Amp MM D
J9 10μl 15μl Amp MM E
J10 10μl 15μl Amp MM E
J11 10μl 15μl Amp MM E
J12 10μl 15μl Amp MM F
J13 10μl 15μl Amp MM F
J17 10μl 15μl Amp MM F
  • Vortexed and spun down samples
  • Placed samples in the thermocycler Genomic PCR program

1X Cleanup

  • After PCR, added 25μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
  • Placed tubes on shaker at room temp for 15 minutes
  • Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed supernatant from each tube on the magnet plate without disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
  • Resuspended beads in 16μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed supernatant when clear to new labeled PCR strip tubes

QC

Sample Std 1 Std 2 Avg ng/μl
S1 198 RFU 21757 RFU 67
S2 - - 150
S3 - - 163
S4 - - 134.5
J1 - - 171.5
J2 - - 115.5
J3 - - 165.5
J4 - - 138.5
J5 - - 163
J6 - - 119.5
J7 - - 146
J8 - - 150.5
J9 - - 169.5
J10 - - 162.5
J11 - - 165
J12 - - 141.5
J13 - - 166.5
J17 - - 143

See full report here

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Written on September 25, 2019