GSM5851552: ES_ChIP_Input_3; Mus musculus; ChIP-Seq
ES cells were fixed with 1% formaldehyde in DMEM at 37 °C for 10 min, neutralized in DMEM containing 125 mM glycine at room temperature for 5 min and washed with PBS three times. Fixed cells were collected with 2ml soft lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 0.1% NP-40 10% Glycerol) and centrifuged at 3000 rpm for 15 min. The cell pellet was resuspended in 100µl SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.0), and sonicated. Soluble chromatin was diluted in 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167mM NaCl) and incubated with 2µg anti-Pol II Ser5P (Abcam, ab5131), anti-Pol II Ser2P (Abcam, ab5095) antibodies or IgG at 4 °C overnight. Bound chromatin was purified on magnetic beads after washings in low salt buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100), high salt buffer (20 mM Tris-HCl, pH 8.0 ,2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X-100) and LiCl buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate). Chromatin was eluted in 1% SDS, 100mM NaHCO3 at 65 °C, reverse-crosslinked and was purified by phenol-chloroform extraction. ChIP–seq libraries were prepared using a NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and the library quality was assessed using a 2100 Bioanalyzer (Agilent). Samples were paired-end sequenced at PE150 on an Illumina Hiseq 4000.