title: GSM8822423: FT190,PLKO,BRCA1 CUT&RUN; Homo sapiens; OTHER
library_construction_protocol: Experiments for CUT&Tag was performed using the CUT&Tag-IT™ R-loop Assay Kit (Active Motif, 53167) according to the manufacturer's protocol. FT190 control and SMARCA4 knockdown cells were seeded overnight, and 200,000 cells per condition were harvested, washed, and immobilized on Concanavalin A-coated magnetic beads. Cells were treated with RNase A or water (control) for 30 minutes at 37°C, followed by incubation overnight at 4°C with either the Active Motif DNA-RNA hybrid (Clone S9.6) primary antibody or BRCA1 antibody (Millipore, cat: OP92). The following day, a rabbit anti-mouse secondary antibody was added, followed by binding of pAG-Tn5 Transposome for targeted tagmentation at R-loop-enriched regions (S9.6) or BRCA1-enriched regions. DNA was extracted and libraries were prepared using indexed primers according to the kit instructions. The libraries were PCR-amplified, purified, and quality-checked using a Bioanalyzer. Sequencing was performed on the NextSeq 2000 using paired-end 100 bp reads.