Approximately 2 × 105 cells were used (~100 μg of chromatin) for histone mark IP, and 1 mg of chromatin was used for BAZ1B-FLAG IP. Cells were fixed with phosphate-buffered saline, containing 1% formaldehyde (Sigma-Aldrich), for 10 min to cross-link proteins and DNA, when the reaction was then stopped by adding 125 mM glycine for 5 min. Cells were lysed with SDS buffer containing 100 mM NaCl, 50 mM tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0), and 10% SDS, at which point chromatin pellets were resuspended in IP buffer containing 1 volume of SDS buffer and 0.5 volume of Triton dilution buffer [100 mM tris-HCl (pH 8.5), 5 mM EDTA (pH 8.0), and 5% Triton X-100]. Chromatin was then sonicated using the S220 Focused-ultrasonicator (Covaris) to generate <300 bp DNA fragments (for histone mark IPs) or the Branson Digital Sonifier to generate 500 to 800 bp DNA fragments (for BAZ1B-FLAG IP).

Sonicated chromatin was incubated overnight at 4°C with primary antibodies [H3K27ac (Abcam), H3K4me1 (Abcam), H3K4me3 (Abcam), H3K27me3 (Cell Signaling Technology), and FLAG (Sigma-Aldrich)] and then for 3 hours with Dynabeads Protein G (Thermo Fisher Scientific). Beads were washed three times with low-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8.0), and 150 mM NaCl] and once with high-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM tris-HCl (pH 8.0), and 500 mM NaCl]. Immunocomplexes were eluted in decross-linking buffer (1% SDS and 100 mM NaHCO3) at 65°C for 2 hours. DNA was purified using QIAquick PCR columns (QIAGEN) and quantified with a Qubit dsDNA HS assay kit (Thermo Fisher Scientific). DNA libraries were prepared by the sequencing facility at European Institute of Oncology according to the protocol described by Blecher-Gonen and colleagues (53), and DNA was sequenced on the Illumina HiSeq 2000 platform. For the FLAG ChIP, samples were run in duplicate.

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