GSM5171466: SgRNA-seq sgRNA_GM_LargeScreen_Rep4; Homo sapiens; OTHER
Up to 500,000 FACS-isolated immortalized cell line cells were processed for scATAC-seq. Cells were pelleted in a fixed-angle rotor at 300 RCF for 5 minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 100 uL of ice-cold lysis buffer (RSB containing 0.1% Tween-20, 0.1% NP-40, and 0.01% digitonin) and incubated on ice for 3-5 minutes. To dilute lysis, 1 mL of chilled RSB-T was added to each tube and the cells were pelleted as before. The supernatant was removed and the pelleted nuclei were resuspended in Diluted Nuclei Buffer (10x Genomics). The nuclei stock concentration was determined for each cell line using Trypan Blue and a total of 5,000 nuclei from each cell line were pooled together and loaded into the 10x Genomics scATAC-seq (v1) transposition reaction. In brief, after the final scATAC-seq libraries were prepared (~150nM final concentration in 20uL ddH2O), 2.5uL of the libraries were used as input for a targeted sgRNA linear amplification PCR reaction using a 5' biotinylated, sgRNA-specific primer (oSP2053: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTaagtatcccttggagaaccaccttg-3') for 25 cycles. PCR product was pooled and purified using a Qiagen Minelute kit (Qiagen). Biotinylated sgRNA fragments were then enriched using Streptavidin MyOne C1 beads (ThermoFisher) and re-suspended in 40uL ddH2O, which was used as input for an exponential PCR amplification reaction for 15 cycles using primers corresponding to P5 (oSP1594: 5'- AATGATACGGCGACCACCGAGA-3') and an indexed P7-containing primer (5'- CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTG-3', where NNNNNNNN is replaced with the index of choice).
GEO Accession
GSM5171466