GSM9377762: Mice-1943, prostate tissue, Pten/Foxa1 Double Knock Out, ,FOXA1(ChIP); Mus musculus; ChIP-Seq
Frozen tissues were ground using agate mortar and pestle and homogenized using the BeadBug benchtop homogenizer. Tissues were then double cross linked with 2mM DSG for 10min, followed by 1% formaldehyde for 10min, at room temperature. Crosslinking was quenched with 0.125 M glycine for 5min at room temperature. Chromatin was fragmented using an E220 focused ultrasonicator (Covaris), 50 ng of Drosophila chromatin was added to each sample as spike-in DNA, and samples were pre-cleared with protein A agarose beads (Millipore) for 1hr. Then samples were incubated with the following antibodies overnight at 4C with rotation: FOXA1 ab23738 (Abcam) and H3K27ac CST 8173S. 1 ug drosophila H3 antibody (Active motif Cat# 61686) was also added per sample. Then, protein A agarose beads were added and incubated for 2h at 4C, followed by washing the beads with 1×dialysis buffer (2mM EDTA, 50mM Tris-Cl, pH 8.0) twice, and IP wash buffer (100mM Tris-Cl, pH 9.0, 500mM LiCl, 1% NP40, 1% deoxycholate) four times. Finally, protein-DNA complexes were eluted (50mM NaHCO3, 1% SDS), crosslinks were reversed, and DNA was purified using DNA Clean & Concentrator-5 kit (ZYMO Research). Total 10 ng ChIP DNA was used to prepare libraries using NEBNextUltra II DNA Library Prep Kit for Illumina (NEB E7645L) according to manufacturer's instructions.