Kun:LabNotes/SingleCellExpr/2011-7-5

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Making RNAseq libraries from 100+ cells[edit]

  • Use Clonetech's SMARTer Ultra Low RNA kit.
  • Samples:
  1. Positive control (1ng)
  2. Negative control (ddH2O)
  3. YangXu: Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), 20.8ug/ml, use 1ul
  4. YangXu: Pancreatic lineage differentiated from hESC, NGN3- (~50000 cells, purified with Qiagen RNeasy), 12.7ug/ml, use 1ul
  • Will process the following samples later if the first attempt works.
  1. mESC (~50 cells)
  2. mE9.5 (~163 cells)
  3. mE11.5 (~2000 cells)
  4. mE13.5 male(~3000 cells)
  5. mE13.5 female(~3000 cells)

ds-cDNA synthesis[edit]

  Make reaction buffer: 19ul dilution buffer + 1ul RNase Inhibitor => 20ul
  RNA denaturing and annealing:
    Reaction buffer                    2.5ul
    total RNA                            1ul
    3’ SMART CDS Primer II A (12uM)      1ul
    Add ~10ul mineral oil
    72C 3min -> transfer to coldblock
  Prepare master mix:
    5X First-Strand Buffer                       1ul
    100mM DTT                                 0.25ul
    dNTP Mix                                     1ul
    SMARTer II A Oligonucleotides (12uM)         1ul
    RNase Inhibitor                           0.25ul
    SMARTScribe Reverse Transcriptase (100U)     1ul
  Add 5.5ul master mix to each reaction, mix by gentle pipetting, spin briefly.
    42C 90min -> 70C 10min
  Purify the ds-cDNA with AmpPure bead: 
    Add 25ul AmpPure beads, mix with pipetting, incubate at RT for 8min;
    Briefly spin-down to separate mineral oil/aqueous; 
    Place the tubes on the magnetic separation device for 5min;
    Pipette out the solution, spin briefly;
    Place the tubes back to the magnetic separation device for 2min;
    Remove the rest of liquid;

PCR amplification[edit]

  Set up QPCR reactions with Kapa HiFi master mix.
    2X Master Mix        25ul
    IS PCR primer (12uM)  2ul
    H2O                  23ul
  95C 2min -> (98C 20sec -> 65C 15sec -> 72C 2min) x 12 cycles (10 cycles for YangXu's samples) -> 72C 3min.
  Purify the dsDNA with AmpPure bead: 
    Add 90ul AmpPure beads, mix with pipetting, incubate at RT for 8min;
    Place the tubes on the magnetic separation device for 5min;
    Pipette out the solution, wash with 200ul 80% EtOH, repeat wash one more time
    Seal tubes and briefly spin down, remove the rest of liquid;
    Air-dry 3-5min;
    Resuspend in 12ul purification buffer; take out 10ul (1ul for Qubit), 1ul (left in the tube) for gel check

PCR products[edit]

TBE PAGE: 1ul; 1ul low mass ladder, 0.5ul ladder, PC, NC, N+, N-

File:7.5.11 smarter.jpg

File:7.5.11 smarter overexposure.jpg

Qubit dsDNA HS assay (1ul for concentration, 9ul left in tube):
PC: 0.333ug/ml
NC: too low
N+: 1.59ug/ml
N-: 1.13ug/ml
IS PCR primer: AAGCAGTGGTATCAACGCAGAGT

Illumina library construction[edit]

  • Covaris shearing to 200bp.
  • End-repair
  • A-tailing
  • TA-ligation
  • Enrichment PCR with Kapa HiFi mastermix, and barcoded primers
  • Mix and size selection.