GSM8605978: MDA-PCa-2b, SMARCA2/4 degrader, SMARCA4, rep2; Homo sapiens; ChIP-Seq
Cells (10 x 10^6) were crosslinked by 10-minutes of 1% formaldehyde. Formaldehyde was quenched by the addition of glycine. Nuclei were isolated with ChIP lysis buffer (1% Triton x-100, 0.1% SDS, 150 mM NaCl, 1mM EDTA, and 20 mM Tris, pH 8.0). Nuclei were sheared with Covaris sonicator (E220) using the following setup: Fill level – 10, Duty Cycle – 15, PIP – 350, Cycles/Burst – 200, Time – 8-minutes). Sheared chromatin was immunoprecipitated overnight with the following antibodies: SMARCA4 (ab110641), FOSL1 (PA5-66880 – Invitrogen) and TEAD1 (BD #610923). Antibody chromatin complexes were pulled down with Protein A or Protein A/G magnetic beads and washed twice in IP wash buffer I. (1% Triton, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in IP wash buffer II (1% Triton, 0.1% SDS, 500 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in IP wash buffer III. (0.25 M LiCl, 0.5% NP-40, 1mM EDTA, 20 mM Tris, pH 8.0, 0.5% NaDOC) and once in TE buffer (10 mM EDTA and 200 mM Tris, pH 8.0). DNA was eluted from the beads by vigorous shaking for 20-minutes in elution buffer (100mM NaHCO3, 1% SDS). DNA was de-crosslinked overnight at 65C and purified with MinElute PCR purification kit (Qiagen). Sequencing ready libraries were generated using NEBNext ® Ultra™ II DNA Library Prep Kit for Illumina ® (New England Biolabs E7645). Libraries were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and profiled using the D5000 ScreenTape on TapeStation 4200 (Agilent Technologies). Libraries were pooled and sequenced on an Illumina sequencer to generate 25 million paired end 50-base pair reads per library.