title: GSM8143664: H3K27ac, β3-AR-stimulated H2A.Z KD BAs, rep2; Mus musculus; ChIP-Seq
library_name: GSM8143664
library_construction_protocol: Adipocytes on plates were crosslinked with 1% formaldehyde at room temperature for 10 minutes, quenched with glycine, and washed twice with ice-cold PBS. Crosslinked adipocytes were then scraped down in PBS supplemented with 0.1% Triton X-100 and 1x protease inhibitor cocktail, followed by incubation on ice for 10 minutes. After centrifugation, the cells were resuspended in PBS supplemented with 0.1% Triton X-100, 1x protease inhibitor cocktail, and 1 mM CaCl2. An appropriate amount of MNase was added to the cell suspension, and MNase digestion was carried out at 37°C for 10 minutes with gentle shaking in a thermomixer. The reaction was quenched by placing the samples on ice, adding 5 mM EDTA and 5 mM EGTA, and centrifuging at 13,000 rpm for 10 minutes at 4°C. The chromatin digestion profile was determined by electrophoresis, and the optimal digestion condition resulted in around 70% of mono-nucleosome. The chromatin was resuspended in ChIP buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM EDTA, 1mM PMSF, 1x protease inhibitor cocktail), rotated at 4°C for 2 hours, and centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatants containing mono-nucleosomes were collected, and the 5 μg of the indicated antibodies (H2A.Z, H3K4me3, H3K27ac, Ribbit IgG) were individually added. Chromatin with each antibody was incubated overnight at 4°C. On the next day, 20 μl DynabeadsTM Protein G beads (ThermoFisher Scientific) were added and incubated for 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 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 RNaseA (10 mg/ml, ThermoFisher Scientific) and incubating at 37°C for 1 hour, followed by supplementation with 3 μl Proteinase K (20 mg/ml, ThermoFisher Scientific) and incubation at 65°C for 3 hours or overnight. Genomic DNA was purified using AMPure XP beads (Beckman Coulter) and dissolved in 20 μl H2O for ChIP-seq. ChIP library preparation was performed using Ovation Ultralow System V2 (Tecan Life Sciences) according to the manufacturer's protocol. 2 ng ChIP DNA was used for library preparation.