Mission human genome shrna library

The cell lines used throughout this study were plated at a seeding density of 3 × 10⁵ cells per well of a six-well culture plate and allowed to attach for 24 h. The lentivirus containing small hairpin RNA (shRNA) (Sigma, Mission Human Genome shRNA Library) against the target gene was inoculated in the presence of 8 μg/ml polybrene (Sigma, H9268, lot no. SLBH5907V) containing media. Cells were selected for 72 h using 1 μg/ml puromycin (Sigma, P9620, lot no. 034M4008V) and subsequently used for various experiments. The sequence of shRNAs used in this study is provided in Supplementary Table S1.

Small hairpin RNAs (shRNA) targeting genes SRSF2, CTCF, DNMT3A, and HIF1α were obtained from Sigma’s Mission Human Genome shRNA Library. Lentivirus containing shRNA plasmid were generated by transfecting HEK293T cells with Δ8.9 and VSVG plasmid along with the required shRNA containing plasmid in 1:0.5:2 ratio using polyethylenimine (PEI). The MCF7 and HCC1806 cell lines were seeded in six-well culture plates, and transduced with a lentivirus solution containing the specific shRNA and polybrene. The cells were then spun at 2200 rpm for 90 minutes, and selected using 1 μg/ml puromycin for 3 days. The cells were then maintained in a humidified incubator at 37°C with 5% CO₂. The knockdown of the targeted genes was confirmed by western blotting, and the cells were then used for downstream experiments. Additional information regarding the reagents used are added in key resources table. The shRNA sequences used are provided in Table S4.