Next-generation sequencing of GeCKO library: H44_1
The genomic DNA was prepared using the Quick-DNA Midiprep Plus Kit (Zymo Research, D4075). Amplification of the gRNA sequences were performed using the NEBNext Ultra II Q5 Master Mix (NEB, M0544L) with the following cycling parameters: 3 min of initial incubation at 98C, 25 cycles at 98C for 10 s, 60 C for 10 s and at 72C for 30 s, and a final elongation step of 2 min at 72C. For each sample, forty separate 50-l reactions with 5 g of genomic DNA in each reaction were performed and then combined. The ~360-bp bands from each sample were purified from DNA-PAGE, quantified, mixed, and sequenced using NextSeq 500 (Illumina) at the The Single Cell & Transcriptomics Core of Johns Hopkins University.