GSM6127805: SR-rep3-gRNA1gRNA2 [mTcell.CP.SR3.G12]; Mus musculus; OTHER
Genomic DNA extraction was performed using TIANamp Genomic DNA kit (TIANGEN) and finally resuspended into 50μl nuclease free water. The DNA concentration was maesured via Q-bit. The SR sgRNA library readout was produced using direct PCR, whereas the TIL sgRNA library readout was produced using two-step PCR. Two regions in the 4-sgRNA tandem cassette were amplified in step-1 PCR using primers specific to the library vector. Region-1 contained sgRNA#1 and sgRNA#2 spacers, Region-2 contained gRNA#2 and sgRNA#3 spacers. Any sequence in the two regions was unique, which could be the indicator for the 4-sgRNA combination. PCR (400~800ng DNA input per reaction, 2~4 reactions per sample) was performed using Ultra II Q5 Master Mix (NEB) with thermocycling parameters as 98oC for 30 sec,  cycles of  (98oC for 10 sec, 60oC for 30 sec, 72oC for 20 sec), 72oC for 2 min. Then, the PCR product was enriched into nuclease free water after size selection via AMPureXP beads. In step-2 PCR and direct PCR, all genomic DNA or step-1 PCR products were amplified with thermocycling parameters as 98oC for 30 sec, cycles of (98oC for 10 sec, 64oC for 30 sec, 72oC for 20 sec), 72oC for 2 min. Then, the redundant adaptor primers were depleted via AMPureXP beads. All PCR cycles were determined by Q-PCR prior to scale-up.