GSM9081692: Input, vehicle-treated control human BAs-rep2; Homo sapiens; ChIP-Seq
For H2A.Z and H3K27ac ChIP-seq in primary human brown adipocytes, cells were washed twice with PBS and then the nuclei were isolated by incubating the cells with lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 1 mM MgCl2, 0.1% NP40, and 1x protease inhibitor cocktail) for 10 minutes on ice. The cell suspension was filtered through a 40 μm strainer, centrifuged at 1,000g for 5 minutes, and washed twice with ice-cold PBS. The nuclei were counted and diluted with PBS to 1 million/ml, then fixed with 1% formaldehyde at room temperature for 10 minutes, quenched with glycine, and washed twice with ice-cold PBS. Crosslinked adipocytes were scraped in PBS supplemented with 1mM PMSF, pelleted at 3,000 g for 5 minutes at 4°C, and suspended in 300 μl lysis buffer (50 mM Tris pH 7.5, 1% SDS, 0.25% sodium deoxycholate, and 1X protease inhibitor cocktail), followed by incubation on ice for 10 minutes. The volume was then increased to 1.2 ml by adding 900 μl of ChIP dilution buffer (50 mM Tris pH 7.5, 0.1% SDS, 150 mM NaCl, 1.84% Triton X-100, and 1x protease inhibitor cocktail). Chromatin was sonicated using a Branson Sonifier SFX250 (50% amplitude, 3.5 minutes). After centrifugation at 13,000 rpm for 10 minutes, the supernatant was transferred to a new tube and mixed with 1.8 ml ChIP dilution buffer. For each ChIP reaction 1.5 ml lysate was incubated overnight at 4°C with 5 μg of the indicated antibodies antibodies (H2A.Z (Active Motif, 39943), H3K27ac (Active Motif, 39133)).On the next day, 20 μl of DynabeadsTM Protein G beads were added and incubated for additional 2 hours at 4°C. The beads were washed twice with ice-cold ChIP buffer with 0.1% SDS, washed twice with ice-cold High-salt wash buffer (50 mM Tris pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM EDTA, 0.1% SDS), washed twice with ice-cold LiCl buffer (10 mM Tris pH 8.0, 0.25 M LiCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 1 mM EDTA), and washed once with ice-cold TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). The protein-DNA complex was eluted from beads by adding 100 μl of freshly prepared elution buffer (10 mM Tris pH 8.0, 0.1% SDS, 150 mM NaCl, 5 mM DTT) and incubated at 65°C in a thermomixer set at 600 rpm for 1 hour. Then, reverse crosslinking was carried out by adding 2 μl of RNaseA (10 mg/ml, ThermoFisher Scientific, EN0531) and incubating at 37°C for 1 hour, followed by supplementation with 3 μl of Proteinase K (20 mg/ml, ThermoFisher Scientific, 25530049) and incubation at 65°C for 3 hours or overnight. Genomic DNA was purified using AMPure XP beads (Beckman Coulter, A63881) and dissolved in 20 μl H2O. ChIP library preparation was performed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) according to the manufacturer's protocol. 5 ng ChIP DNA was used for library preparation.