GSM4976567: PolII-3T3-L1-CTL; Mus musculus; ChIP-Seq
3T3-L1 preadipocytes with indicated treatments were used for ChIP-Seq analysisFor ChIP with Pol II (Bethyl, A300-653A) andC/EBPβ (Santa Cruz, sc-150) antibodies, cells were fixed with 1% formaldehyde for 15 min at RT. For ChIP with HA antibody (Abcam, ab9110), cells were fixed with disuccinimidyl glutarate (DSG) (5 mM) for 45 min at RT, washed twice with PBS and fixed with 1% formaldehyde for another 15 min at RT. Fixation was stopped by 0.14 M glycine at RT for 10 min, followed by two washes with PBS. For each ChIP, 1×107 cells were resuspended in Lysis Buffer A (50 mM Tris-cl, pH7.8, 10 mM EDTA, 1% SDS ) supplemented with protease inhibitors (Roche). Chromatin was fragmented to 150-300 bp on a Bioruptor for 30 cycles of 30 s on and 30 s off. Chromatin lysates were then diluted 10-fold with Buffer D (20 mM Tris, pH7.8, 150 mM NaCl, 2 mM EDTA and 1% Triton X-100) supplemented with protease inhibitors, incubated with 5 μg anti-HA (Abcam, ab9110), 5 μg anti-C/EBPβ (Santa Cruz, sc-150), 5 μg anti-Pol II (Bethyl, A300-653A)antibodies overnight at 4oC, and then immunoprecipitated with Protein G magnetic beads (Biorad, 161-4023) with rotation at 4oC for 4 hr. Immunoprecipitations were washed once with Buffer E (20 mM Tris, pH8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS and 1% Triton X-100) for 15 min at 4oC, once with Buffer E containing 400 mM NaCl for 15 min at 4oC, and twice with Buffer F (50 mM Tris, pH8.0, 1 mM EDTA). Formaldehyde crosslinked chromatin was reversed in Buffer G (50 mM Tris, pH8.0, 10 mM EDTA, 1% SDS) at 65oC overnight. DNA was isolated using Qiagen PCR product purification kit (#28104). Library was generated using a KAPA Hyper Prep Kit (Roche, KK8503) following manufacturer's instructions. Adaptors and primer sequences from Roche were used for library construction and amplification. After PCR amplification, the 250-450 bp fragments were purified using KAPA pure beads (Roche, KK8000) following manufacturer's instructions.