GSM9499521: ChIP-gH2AX_S7HY-c7; Mus musculus; ChIP-Seq
cells were crosslinked with 1% formaldehyde for 10 min at room temperature and then quenched with 0.2 M glycine for 5 min and washed once with cold PBS. Nuclei were resuspended in 300 μl Lysis buffer 2 (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0, and cOmplete protease inhibitors) and incubated on ice for 30 min. Chromatin was sonicated to an average fragment size range of 300-500bp and subsequently centrifugated at 12,000 x g at 4°C for 15 min. Supernatants were diluted with Dilution Buffer (1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and cOmplete protease inhibitors). ChIP-grade protein A/G magnetic beads (Pierce, 26162) were conjugated for at least 3 hours in 1X PBS and 0.05% NP-40 with anti-H2A.X antibody (ab20669, Abcam), or anti-ɣH2AX (Sigma, 05-636-I). Chromatin samples and antibody-conjugated magnetic beads were incubated overnight at 4°C on a rotator. Magnetic beads were then washed 3 times with ice-cold RIPA buffer (1% NP-40, 0.7% Na-deoxycholate, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 M LiCl2, and complete protease inhibitors) and once with Buffer TE (0.1 mM EDTA, 10 mM Tris-HCl pH 8.0). Magnetic beads and input samples (1% of total chromatin) were resuspended in freshly prepared RC buffer (0.1 M NaHCO3 and 1% SDS) in the presence of proteinase K for 5 hours at 60°C on a thermomixer with 900 rpm. DNA was purified using a Binding Buffer NTB and Nucleospin PCR Clean-up Kit from Macherey-Nagel. library preparation was performed using NEBNext® Ultra II DNA Library Prep Kit for Illumina® (NEB) according to manufacturer instructions.