GSM1402782: mH2A1_HepG2_ChIPseq; Homo sapiens; ChIP-Seq
Formaldehyde was added to the culture medium to a final concentration of 1%. Cross-linking was allowed to proceed for 10 min at room temperature and stopped by addition of glycine at a final concentration of 0.125 M, followed by an additional incubation for 5 min. Fixed cells were washed twice with TBS (20 mM Tris at pH 7.4, 150 mM NaCl) and harvested in SDS Buffer (50 mM Tris at pH 8.1, 0.5% SDS, 100 mM NaCl, 5 mM EDTA, and protease inhibitors). Cells were pelleted by centrifugation, and suspended in 4 mL of IP Buffer (100 mM Tris at pH 8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA). Cells were disrupted by sonication. The lysate was then diluted with IP buffer to a final volume of 9 mL. For each immunoprecipitation, diluted lysate was precleared by addition of blocked protein A beads. Samples were immunoprecipitated overnight at 4°C with polyclonal antibodies specific for either macroH2A1 (3 μg), macroH2A2 (3 μg), IgG (3μg, ab46540, Abcam). Immune complexes were recovered by adding 30 μL of blocked protein A beads and incubated for 2 h at 4°C. Beads were washed and eluted, and cross links were reversed as described by Aparicio (1999). This included successive washes in 1 mL of Mixed Micelle Buffer (20 mM Tris at pH 8.1, 150 mM NaCl, 5 mM EDTA, 5% w/v sucrose, 1% Triton X-100, and 0.2% SDS), Buffer 500 (50 mM HEPES at pH 7.5, 0.1% w/v deoxycholic acid, 1% Triton X-100, 500 mM NaCl, and 1 mM EDTA), LiCl Detergent Wash Buffer (10 mM Tris at pH 8.0, 0.5% deoxycholic acid, 0.5% NP-40, 250 mM LiCl, and 1 mM EDTA), and TE (pH 7.5). The eluted material was phenol/chloroform-extracted and ethanol-precipitated. DNA was resuspended in 50 μL of waternand quantified with PicoGreen. Libraries were prepared for sequencing using standard Illumina protocols
GEO Accession
GSM1402782