Kun:LabNotes/SingleCellExpr/2011-7-5

From ZhangLabWiki
Jump to navigation Jump to search

Making RNAseq libraries from 100+ cells

  • Use Clonetech's SMARTer Ultra Low RNA kit.
  • Samples:
  1. Positive control (1ng)
  2. mESC (~50 cells)
  3. mE9.5 (~163 cells)
  4. mE11.5 (~2000 cells)
  5. mE13.5 male(~3000 cells)
  6. mE13.5 female(~3000 cells)
  7. YangXu: Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), use 1/10
  8. YangXu: Pancreatic lineage differentiated from hESC, NGN3- (~50000 cells, purified with Qiagen RNeasy), use 1/10

ds-cDNA synthesis

    total RNA                          3.5ul
    3’ SMART CDS Primer II A (12uM)      1ul
    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;
    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

  Set up QPCR reactions with Kapa HiFi master mix.

Illumina library construction