title: GSM8143709: β3-AR-stimulated H2A.Z KD BAs, replicate1, pilot; Mus musculus; OTHER
library_name: GSM8143709
library_construction_protocol: Micro-C was performed using  DovetailTM Micro-C kit (Dovetail GENOMICS) according to the manufacturer's protocol. Vehicle-treated control, β3-AR-stimulated control, and β3-AR-stimulated H2A.Z KD brown adipocytes were washed twice with room temperature PBS and then scraped down in 1 ml PBS per well of a 6-well plate. The suspended adipocytes were crosslinked with 0.3 M DSG at room temperature for 10 minutes with rotation, followed by 1% formaldehyde at room temperature for 10 minutes with rotation. Adipocytes were collected with a spin at 3,000 g for 5 minutes and washed with 1x Wash buffer twice. Crosslinked adipocytes from a well of a 6-well plate were resuspended in 200 μl 1x Nuclease Digest buffer and divided into 4 tubes to determine the appropriate amount of Micrococcal nuclear (MNase) by incubating at 22°C for 15 minutes in an agitating thermal mixer set at 1,250 rpm. The MNase reaction was inactivated by adding 5 μl of 0.5M EGTA and 3 μl of 20% SDS, followed by incubation at 22°C for 5 minutes in an agitating thermal mixer set at 1,250 rpm. The fragment size distribution was assessed using a Bioanalyzer HS DNA chip. The optimal digestion condition resulted in 40-70% of mono-nucleosome. MNase-digested chromatin was then subjected to proximity ligation. Library preparation, ligation capture and amplification were performed using the DovetailTM Micro-C kit (Dovetail GENOMICS) according to the manufacturer's protocol.