GSM5949840: mESC_BRM014_2h_ChIP-seq_BRG1_rep2; Mus musculus; ChIP-Seq
After the indicated treatment interval, cells were fixed for 1 h in 2 mM DSG with the addition of 1% formaldehyde for the final 12.5 min, as described in.86 Crosslinking was quenched by the addition of glycine to a final concentration of 0.125 M. Cells were collected and washed with ice-cold 1X PBS before being resuspended in Sonication Buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 0.5 mM EGTA, 1X Complete Mini EDTA-free Protease Inhibitor Cocktail [Roche 11836170001], 0.5% SDS, and 0.5 mM PMSF) at a concentration of 1 x 108 cells per mL. Chromatin was sheared to an average fragment size of ∼200 bp using a QSonica sonicator, flash-frozen in liquid nitrogen, and stored at -80°C until use. ChIP material was diluted into IP buffer (20 mM Tris pH 8.0, 2 mM EDTA, 0.5% Triton X-100, 150 mM NaCl, 10% Glycerol, 5% BSA) and pre-cleared with 30 μL of Protein A agarose beads (Millipore Cat No. 16-125) for 1 h at 4°C. Cleared samples were collected and combined with primary antibody before overnight incubation at 4°C with rotation. Subsequently, 200 μL Protein A beads were added to each IP reaction, and samples were rotated for 2 h at 4°C. Samples were washed once with Low-Salt Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.1% SDS), three times with High-Salt Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 500 mM NaCl, 0.1% SDS), once with Lithium Chloride Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 250 mM LiCl, 1% IGEPAL CA-630, 1% sodium deoxycholate), and twice with TE Buffer (10 mM Tris-HCl, 1mM EDTA). Each wash was performed by rotating samples for 3 minutes with 1 mL volume of ice-cold wash solution. Two elutions were performed by resuspending beads in Elution Buffer (100 mM NaHCO3, 1% SDS) and rotating for 15 min at room temperature (22°C). The combined eluate was supplemented with 200 mM NaCl and incubated overnight in a 65°C water bath. Samples were treated with Proteinase K (New England Biolabs P8107), extracted with phenol:chloroform:isoamyl alcohol (25:24:1, Sigma P3803), and resuspended in 65 μl H2O. To enable accurate sample normalization, an equal amount of fragmented D. melanogaster DNA was added to the eluate of each sample. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions.