GSM9081711: FSK-treated control human BAs, rep1; Homo sapiens; OTHER
Micro-C was performed using  DovetailTM Micro-C kit (Dovetail GENOMICS) according to the manufacturer's protocol. Vehicle-treated control, FSK-treated control, and FSK-treated H2A.Z KD human brown adipocytes 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, followed by fixation with 3 mM DSG at room temperature for 35 minutes and 1% formaldehyde at room temperature for 10 minutes with rotation. The fixation was quenched by adding Tris-HCl, pH7.5 to a final concentration of 375 mM. Nuclei were collected with a spin at 2,000 g for 5 minutes and washed twice with ice-cold PBS. Nuclei pellet was resuspended with MNase digestion buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.2% NP40, and 1x protease inhibitor cocktail) and appropriate amount of MNase was added to digest genomic DNA at 37°C for 20 minutes in an agitating thermal mixer set at 1,000 rpm. The MNase reaction was inactivated by adding 0.5 M EGTA to a final concentration of 4 mM, followed by incubation at 65°C for 10 minutes in an agitating thermal mixer set at 1,000 rpm. The fragment size distribution was assessed using a Bioanalyzer or Tapestation HS DNA chip. The optimal digestion condition resulted in 40-70% of mono-nucleosomes. MNase-digested chromatin was then subjected to proximity ligation, library preparation, ligation capture and amplification according to the manufacturer's protocol. Library preparation, ligation capture and amplification were performed using the DovetailTM Micro-C kit (Dovetail GENOMICS) according to the manufacturer's protocol.