GSM7984645: BAT, 30C, H3K27ac ChIP, WT1; Mus musculus; ChIP-Seq
The BAT pads were minced in ice-cold PBS and dissociated into single-cell suspensions using the Adipose Tissue Dissociation Kit (Miltenyi Biotec). Collected cell pellets were fixed in 2mM DSG for 30 min and cross-linked with 1% formaldehyde for 10 min. Cross-linking was quenched with addition of 125 mM glycine for 5 min and followed by twice ice-cold 1× PBS washes. Cross-linked cell pellets were re-suspended in ice-cold Shearing Buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS, and protease inhibitor) and sonicated with a Biorupter (UCD300, Cosmo Bio) to shear chromatin to 200-1000bp fragments. Sonicated chromatin lysates were incubated with the antibody against H3K27ac (CST#8173) at 4 °C overnight and then immunoprecipitated with IgG paramagnetic beads (Invitrogen). Chromatin immune complexes were eluted twice in Bead Elution Buffer (0.1 M NaHCO3 and 1% SDS). The eluted chromatin was de-crosslinked overnight in 0.2M NaCl at 65 °C and the associated proteins were digested with proteinase K. The final DNA was purified with the MinElute PCR Purification Kit (Qiagen). Sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to manufacturer's instructions. Library concentrations were quantified by a Bioanalyzer (Agilent)