title: GSM5129583: 3T3-L1-PolII-shCTL-CTL; Mus musculus; ChIP-Seq
library_construction_protocol: 3T3-L1 preadipocytes with indicated treatments were used for ChIP-Seq analysis. For ChIP with anti-ELK1 antibodies, cells were fixed with 5mM DSG for 45 min at RT,then fixed with 1% formaldehyde for 15 min at RT.  For ChIP with anti-Pol II and anti-Pol II-S2P antibodies, cells were fixed with 1% formaldehyde for 15 min at RT. Fixation was stopped by adding glycine to a final concentration of 0.14 M and incubating at RT for 10 min, followed by two washes with cold PBS.    For each ChIP, 1×107 cells were resuspended in Buffer A (50 mM Hepes, pH7.6, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) supplemented with protease inhibitors (Roche), incubated on ice for 10 min, and spun at 1,000 g for 10 min at 4oC. Pellet (nuclear fractions) was washed twice with Buffer B (10 mM Tris, pH7.8, 200 mM NaCl, 1 mM EDTA and 1 mM EGTA) supplemented with protease inhibitors, and resuspended in 0.3 ml Buffer C (50 mM Tris, pH7.8, 10 mM EDTA and 1% SDS) supplemented with protease inhibitors. 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-ELK1 (Abcam, ab32106),  5 μg anti-Pol II (Bethyl, A300-653A), 5 μg anti-Pol II-S2P (Abcam, ab5095), 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, and glutaraldehyde crosslinked chromatin was reversed in buffer G containing 1 mg/ml protease K at 65oC overnight as previously described51. 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.