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===Making RNAseq libraries from 100+ cells=== *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==== Make reaction buffer: 19ul dilution buffer + 1ul RNase Inhibitor => 20ul RNA denaturing and annealing: Reaction buffer 2.5ul total RNA 1ul 3β 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==== 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==== 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==== *Covaris shearing to 200bp. *End-repair *A-tailing *TA-ligation *Enrichment PCR with Kapa HiFi mastermix, and barcoded primers *Mix and size selection.
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