Kun:LabNotes/SingleCellExpr/2011-7-5
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Making RNAseq libraries from 100+ cells
- Use Clonetech's SMARTer Ultra Low RNA kit.
- Samples:
- Positive control (1ng)
- mESC (~50 cells)
- mE9.5 (~163 cells)
- mE11.5 (~2000 cells)
- mE13.5 male(~3000 cells)
- mE13.5 female(~3000 cells)
- YangXu: Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), use 1/10
- 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.