GSM7814539: BAT_CEBPb_5pm_4C_34415; Mus musculus; ChIP-Seq
Interscapular BAT pads (two lobes) were minced and cross-linked in 1X PBS/1% formaldehyde at room temperature for 20 min. The cross-linking was stopped by adding glycine at a final concentration of 125 mM. After 5-min incubation at room temperature, the BAT pieces were washed three times with ice-cold 1X PBS. Cross-linked BAT pieces were suspended in ice-cold ChIP dilution buffer (50 mM HEPES pH7.5, 150 mM NaCl, 1 mM EDTA, 1.1% Triton X-100, 0.11% sodium deoxycholate, 0.1% SDS) with cOmpleteProtease Inhibitor Cocktail (Roche), and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich), and sonicated (3 times for 10 s each with a 60-s interval at 10% amplitude, followed by 3 times for 10 s each with a 60-s interval at 15% amplitude) with probe sonication (Fisher Scientific, FB705 Sonic Dismembrator). Lysates were centrifuged at 20,817 rpm for 15 min at 4°C to remove lipid. After centrifugation, chromatin lysates were brought to 1 ml with ChIP dilution buffer and an aliquot was saved as 5% input sample. After the addition of 50 ml of Pierce ChIP-grade Protein A/G Magnetic Beads (Thermo Fisher Scientific, 26162), samples were incubated at 4°C for 1 h. The precleared samples were divided into 2 tubes and subjected to immunoprecipitation at 4°C overnight with 3-5 mg of antibodies ChIP-seq libraries were prepared from independent formaldehyde-crosslinked biological replicates of interscapular BAT from dissected from independent mice. Replicates were pooled on a PCR cleanup column prior to sequencing library generation. Libraries were generated according to Illumina ChIP-seq guide, utilizing Illumina adapter oligos and primer sequences for all library construction and amplification steps. Enzymes for library generation were from New England Biolabs. Size selection of adaptor ligated DNA was performed prior to PCR library amplification using Agencourt AMPure XP Beads (Beckman Coulter, A63881) and subsequently purified with a MinElute PCR Purification kit (QIAGEN, 28004). Next-generation sequencing was performed utilizing with the Illumina HiSeq2000.