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:
- Positive control (1ng)
- Negative control (ddH2O)
- YangXu: Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), 20.8ug/ml, use 1ul
- 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.
- mESC (~50 cells)
- mE9.5 (~163 cells)
- mE11.5 (~2000 cells)
- mE13.5 male(~3000 cells)
- 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 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.