GSM5911732: iBAT ZT_03; Mus musculus; ChIP-Seq
ChIP was performed as previously described (Mir AA et al., Methods Mol Biol. 2019). Briefly, iBAT samples pooled from eight mice (approx. 250-350 mg combined; experiment 1) were homogenized in lysis buffer (10 mM HEPES-KOH, pH 7.3, 10 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 1× cOmplete™ Protease Inhibitor (Roche Diagnostics, Almere, The Netherlands)), filtered through a Falcon® 70 µm cell strainer (Life Sciences, Corning, New York, USA), cross-linked in 10 mL 1% formaldehyde in PBS for 15 minutes and quenched with 1.5 mL 1 M glycine for 5 minutes. After washing, pelleted nuclei were further lysed twice by resuspension in 1 mL cold fast IP buffer (50 mM NaCl, 50 mM Tris–HCl (pH 7.5), 5 mM EDTA, 0.5% v/v NP40, 1% v/v Triton X-100, 1× cOmplete™ Protease Inhibitor) and passing through a 24G syringe. Chromatin was subsequently sheared (Bioruptor® plus, Diagenode, Liège, Belgium) in 1 mL shearing buffer (1% v/v SDS, 10 mM EDTA, 50 mM Tris–HCl (pH 8), 1× cOmplete™ Protease Inhibitor). Sheared chromatin was then centrifuged, and 10% of each sample was stored at -20°C to be used as input. The chromatin was diluted in 7.2 mL dilution buffer (0.01% v/v SDS, 1.1% v/v Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl (pH 8.0), 0.167 M NaCl, 1× cOmplete™ Protease Inhibitor) and incubated overnight at 4°C with 8 µg of PPARγ antibody (2443, Cell Signaling Technology, Danvers, Massachusetts, USA). IP was cleared by centrifugation, and the top 90% was incubated with 100 µL bovine serum albumin-blocked DynaBeads™ (11203D; Thermo Fisher Scientific, Waltham, USA) for 6 hours at 4°C. Unbound complexes were removed by washing with fast IP buffer. Beads were collected and washed in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0), and ChIP'ed chromatin was collected by elution with 100 µL beads elution buffer (0.105 M NaHCO3, 1% SDS) for 15 minutes at room temperature two times. Both the input sample and the ChIP'ed chromatin were de-cross-linked by addition of 4 µL and 8 µL 5 M NaCl, respectively, followed by overnight incubation at 65°C. Samples were subsequently treated with RNAse A and Proteinase K, and DNA was purified using MinElute® columns from the PCR Purification Kit (QIAGEN, Hilden, Germany) using the manufacturer's protocol. A stranded library (fragment selection 100-500bp) was constructed for 100bp paired-end sequencing on BGI's platform.