GSM9041534: In-vitro CRISPR Screen of Epigenetic Regulators in the treatment naïve WND238 Cells; Homo sapiens; OTHER
Post-treatment WND238 cells were trypsinized, washed, and processed with Dead Cell Removal Kit (Miltenyi Biotec) following manufacturer's instructions. Genomic DNA (gDNA) extracted from 1 million cells using Blood & Cell Culture DNA Mini Kit (Qiagen cat. no. 13323). Genomic DNA with ≥1000× coverage (6.7 µg) was extracted, quantified, and used for indel amplification of integrated gRNAs via 26–28 cycles of PCR using NEBNext Q5 Hot Start HiFi Master Mix and CROPseq-NGS-Fwd/Rev primers. PCR products (expected size: 291–298 bp) were validated by agarose gel electrophoresis, purified using the Monarch DNA Gel Extraction Kit, pooled, and sequenced on a NextSeq 1000 (Illumina) with a P1 100-cycle kit (dual indexing, single-end; 8 bp × 8 bp × 120 bp).