GSM8309479: RNAPII 16h Ser2 DMSO ChIP-Seq; Homo sapiens; ChIP-Seq
Cells were fixed in 1 % formaldehyde for 10 min at room temperature. Then, glycine was added to a final concentration of 0.125 M and the reaction was incubated for 5 min at room temperature. Fixed cells were washed twice with ice-cold PBS, and lysed for 5 min in ice-cold ChIP cell lysis buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5 % NP-40 alternative, protease inhibitors). Nuclei were pelleted by centrifugation at 3900 g for 5 min at 4ºC, and lysed for 5 min in ChIP nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS, protease inhibitors) and incubated 5 minutes on ice. Lysates were sonicated using VCX130 (Sonics & Materials, Inc) with the following settings: 8 cycles, 30 % Amp, 15 s ON, 25 s OFF. Sonicated chromatin was centrifuged at 17000 g at 4ºC for 10 min and the supernatant was stored at -80ºC until immunoprecipitation. Before immunoprecipitation, 15 µl of Dynabeads Protein A was washed twice with 900 µl 5 mg/ml BSA in PBS and reacted for 1 h or more with 3 µg of the corresponding antibody (Phospho-Histone H2A.X: ab2893, Abcam; H3: ab1791, Abcam) in 500 µl of 5 mg/ml BSA in PBS at room temperature. The beads were washed again with 900 µl 5 mg/ml BSA in PBS before use. The concentration of sonicated chromatin was quantified by using Quick Start Bradford protein 1x dye reagent (Bio-Rad). 50 µl chromatin were used as Input. 0.6 mg of chromatin was 1:5 diluted with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, protease inhibitors), and incubated with prepared beads overnight at 4ºC with rotation. Chromatin bound to beads was washed twice in ChIP low salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), twice in ChIP high salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl),  twice in ChIP LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 % NP40 alternative, 1 % sodium deoxycholate, 1 mM EDTA), and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Most of the TE buffer was removed after the beads were centrifuged at 3000 rpm for 3 min and put on Magnet. The remaining TE was removed after the beads were centrifuged again at 13200 rpm for 1 min and put on Magnet. The beads were resuspended in 50 µl elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS) and incubated at 65°C for 10 min followed by centrifugation at 12000 rpm for 1 min. The supernatant was transferred to a new DNA low binding tube (Sarstedt) and incubated at 65°C overnight for de-crosslinking. Reverse cross-linked supernatant was mixed with 50 µl of TE and incubated with RNase A at 37°C for 1 h. The supernatant was further incubated with 0.7 mg/ml proteinase K at 55°C for 2 h. DNA was purified by DNA Clean & Concentrator-5 and eluted with 8 µl EB (10 mM Tris-HCl pH 8.5). The purified DNA was quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). 1 ng of DNA was used to construct DNA library according to Illumina's Nextera XT DNA Library Preparation Kit protocol. After being cleaned up, indexed libraries were quantified, normalized and pooled, sequenced on Illumina NextSeq system NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads following manufacturer's protocol single end on 4 lanes (L1-L4)