SMARCA4 Chromatin immunoprecipitation-sequencing (ChIP-Seq) and analysis

H1299 cells stably transduced with pINDUCER20-SMARCA4 vector were treated with doxycycline to induce expression of SMARCA4 for 5 days or left untreated. Cells were then treated with IACS-10759 (100nM) for 24 hours, cells were then harvested and prepared for ChIP-Seq. ChIP assays were performed as described in⁵⁵ with minor modifications. Briefly, ~8×10⁷ cells were harvested by cross-linking with 1% (wt/vol) formaldehyde for 10min at 37^oC with shaking. After quenching with 150mM glycine for 10min at 37^oC with shaking, cells were washed twice with ice-cold PBS and frozen at −80^oC for further processing. Cross-linked pellets were thawed and lysed on ice for 30min in ChIP harvest buffer (12mM Tris-Cl, .1x PBS, 6mM EDTA, .5% SDS) with protease inhibitors (Sigma). Lysed cells were sonicated with a Bioruptor (Diagnode) to obtain chromatin fragments ~200 to 500bp and centrifuged at 15,000g for 15min to obtain a soluble chromatin fraction. In parallel with cellular lysis and sonication, SMARCA4 antibody (Abcam: ab110641) at 5ug/3×10⁶ cells was coupled to 30ul of magnetic protein G beads in binding/blocking buffer (PBS+.1%TWEEN+.2% BSA) for 2h at 4^oC with rotation. Soluble chromatin was diluted 5x using ChIP dilution buffer (10mM Tris-Cl, 140mM NaCl, .1% DOC, 1% Triton-X, 1mM EDTA) with protease inhibitors and added to the antibody- coupled beads with rotation at 4^oC overnight. After washing, samples were treated with elution buffer (10mM Tris-Cl pH 8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS), RNAase A and Proteinase K and cross-links were reversed overnight. ChIP DNA was purified using AMPure XP beads (Agencourt) and quantified using the Qubit 2000 (Invitrogen) and Bioanalyzer 1000 (Agilent).