GSM5277021: WT_rep2; Homo sapiens; RNA-Seq
For RNA-seq, the RNA was extracted with HiPure Total RNA Mini Kit (Magen, Cat#R4111-03).For ChIP-seq, the PGP1 cells (~5x106 cells/repeat)  were crosslinked in 1% formaldehyde for 10 min at room temperature with rotation. Cross-linking was stopped by incubating with 0.125M glycine for 5 min at room temperature with rotation.Then cells were collected and treated in  cell lysis buffer (10mM Tris-HCl (pH 8.0), 140 mM NaCl, 0.2% NP-40) plus1x cOmplete Protease Inhibitor (Roche) and incubated on ice for 10 min to isolate nuclei. After washed once with cold DBPS, isolated nuclei were sonicated in Nuclear lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1% SDS) with 1x cOmplete Protease Inhibitor (Roche) in Diagenode Bioruptor Pico to obtain 200-500bp chromatin fragments. The sonication condition was 10 cycles of 20s/on and 30s/off.  After centrifuging, the soluble sonicated fragments were incubated with 5ug antibody and Protein A/G Dynabeads with rotating overnight at 4 °C to enrich fragmented chormatin.Then the DNA fragments were eluted with the elution buffer (100mM NaHCO3 and 1%SDS) and reverse crosslinked at 67 °C over night. Finally, the immunoprecipitation DNA were purified with Phenol-chloroform Protocol for chip-seq. The DNA were sent to Geekgene (Beijing, China) for ChIP-seq library construction and sequencing. For RNA-seq and ChIP-seq , the libraries were prepared for sequencing using standard Illumina protocols.