title: GSM2990419: H3K27me3 ChIP-seq in G34W; Homo sapiens; 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_20. 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 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 μg of antibodies overnight at 4 °C. Protein A/G beads (Millipore) were then added and incubated for 1 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). For ChIP-seq, sequencing libraries were prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer's protocol. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250-450 bp (insert plus adaptor) were band isolated from an agarose gel. DNA fragments were sequenced using single-end sequencing technology on Illumina HiSeq 3000 platform.
attributes.tag: GEO Accession
attributes.value: GSM2990419