title: GSM2934783: J1_shZNHIT1-6_H2AZ ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seq were performed following the protocol: Using ChIP-seq technology to generate high-resolution profiles of histone modifications. O'Geen H, Echipare L, Farnham PJ. Methods Mol Biol. 2011;791:265-86. doi: 10.1007/978-1-61779-316-5_25. ChIP analysis was performed as described previously (Li et al., 2014) with minor modification. In brief, cells were cross-linked with 1% formaldehyde for 10 min and stopped with 125 mM glycine for 5 min. Fixed cells were scraped from dishes and washed with PBS for 2 times. Cell pellets were resuspended with cell lysis buffer (5 mM PIEPES pH 8.0, 85 mM KCl, 1% NP-40) with protease inhibitor cocktail (Roche) and 10 mM phenylmethanesulfonyl fluoride (PMSF). After 20 min rotation in 4 °C, cell nuclei were pelleted by centrifugation. The nuclei were resuspended with nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS) containing protease inhibitors and sonicated using Bioruptor Sonicator (Diagenode). Length of most sheared DNA fragments were between 100-400 base pairs. Sonicated chromatin contains approximately 20-30 μg of DNA were diluted 4 fold with dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.01% SDS) and were subjected to immunoprecipitation with 2–3 μg of antibodies overnight at 4 °C. Protein A/G beads (Millipore) were then added and incubated for 2 hr. The immunoprecipitates were washed twice with low-salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.01% SDS) and high-salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2mM EDTA, 1% Triton X-100, 0.01% SDS) and then once with LiCl buffer (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP40, 1% Na-Deoxycholate). DNA was eluted with vigorously shaking in elution buffer (50 mM NaHCO3, 1% SDS) for 15 min and then reverse crosslink in the presence of 0.3M NaCl at 67 °C for 12 hr. DNA was purified using PCR purification kit (Qiagen) and analyzed by quantitative real-time PCR on the ABI 7500-FAST System using the Power SYBR Green PCR Master Mix (Applied Biosystems). ChIP–seq samples were sequenced using the Illumina Solexa Hiseq 2000 or NextSeq 500.
attributes.tag: GEO Accession
attributes.value: GSM2934783