GSM4972731: Pol2_WT_1; Mus musculus; ChIP-Seq
Fresh-frozen VP tissues from 12-week-old mice were pulverized (Cryoprep Impactor, Covaris), resuspended in PBS + 1% formaldehyde, and incubated at room temperature for 20 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration) for 15 minutes at room temperature, then washing in ice cold PBS + EDTA-free protease inhibitor cocktail (PIC; #04693132001, Roche). Multiple biological replicates were combined for each condition in two distinct pools (replicates). Chromatin was isolated by the addition of lysis buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0), 0.1% NaDOC, 0.13 M NaCl, 1X PIC) + sonication buffer (0.25% sarkosyl, 1 mM DTT) to the samples, which were maintained on ice for 30 minutes. Lysates were sonicated (E210 Focused-ultrasonicator, Covaris) and the DNA was sheared to an average length of ~200-500 bp. Genomic DNA (input) was isolated by treating sheared chromatin samples with RNase (30 minutes at 37°C), proteinase K (30 minutes at 55°C), de-crosslinking buffer (1% SDS, 100 mM NaHCO3 (final concentration), 6-16 hours at 65°C), followed by purification (#28008, Qiagen). DNA was quantified on a NanoDrop spectrophotometer, using the Quant-iT High-Sensitivity dsDNA Assay Kit (#Q33120, Thermo Fisher Scientific). On ice,  AR (2 micrograms, #ab108341, Abcam), FOXA1 (6 micrograms, #ab23738, Abcam), RNA Pol II (4 micrograms, #sc899, Santa Cruz Biotechnology) or H3K27ac (10 microliters, #ab4729, Abcam) antibodies were conjugated to a mix of washed Dynalbeads protein A and G (Thermo Fisher Scientific), and incubated on a rotator (overnight at 4°C) with 5 micrograms (AR, FOXA1, RNA Pol II) or 1.5 micrograms (H3K27ac) of chromatin. ChIP'ed complexes were washed, sequentially treated with RNase (30 minutes at 37°C), proteinase K (30 minutes at 55°C), de-crosslinking buffer (1% SDS, 100 mM NaHCO3 (final concentration), 6-16 hours at 65°C), and purified (#28008, Qiagen). The concentration and size distribution of the immunoprecipitated DNA was measured using the Bioanalyzer High Sensitivity DNA kit (#5067-4626, Agilent). Dana-Farber Cancer Institute Molecular Biology Core Facilities prepared libraries from 2 ng of DNA, using the ThruPLEX DNA-seq kit (#R400427, Rubicon Genomics), according to the manufacturer's protocol; finished libraries were quantified by the Qubit dsDNA High-Sensitivity Assay Kit (#32854, Thermo Fisher Scientific), by an Agilent TapeStation 2200 system using D1000 ScreenTape (# 5067-5582, Agilent), and by RT-qPCR using the KAPA library quantification kit (# KK4835, Kapa Biosystems), according to the manufacturers' protocols; ChIP-seq libraries were uniquely indexed in equimolar ratios, and sequenced to a target depth of 40M reads on an Illumina NextSeq500 run, with single-end 75bp reads.