GSM9416108: P3FM3_Fin_2; Homo sapiens; OTHER
Cell pellets were lysed in 1 ml DNAzol by vigorous pipetting according to the manufacturer's protocol (Molecular Research Center Inc.). DNA was precipitated in 0.5 ml 200-proof ethanol (VWR), followed by a second precipitation in 2.5 ml 200-proof ethanol. Precipitated DNA was pelleted by centrifugation at 20,000 g, washed twice with 75% ethanol, and solubilized in 0.5 ml of water. To minimize PCR amplification bias, sgRNA sequences from each sample were amplified in triplicate and pooled at each step. First-round PCR was performed using 50–100 µg gDNA, Hotstart Taq (Takara), and 1 µM each of CRISPR F1 and CRISPR R1 primers (IDT). Thermal cycling conditions were: 95 °C for 5 minutes; 18 cycles of 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 30 seconds; final extension at 72 °C for 5 minutes. Amplicons from the same sample were pooled, and a second PCR was performed to add sample barcodes and Illumina adaptors using 20 µl of PCR1 and 0.5 µM CRISPR R2 plus barcoded forward primers. Thermal cycling conditions were: 95 °C for 5 minutes; 20 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, 72 °C for 30 seconds; final extension at 72 °C for 5 minutes. Second-round PCR products were pooled at equal concentrations and purified using Ampure XP Beads according to the manufacturer's protocol (Beckman Coulter).