GSM1569575: HMC_L.T.R_H3K9me3; Homo sapiens; ChIP-Seq
Total RNA was extracted by using Qiagen RNA extraction kit according to manufacturer’s manual, and then 10ug total RNA was used for downstream library construction. For ChIP asssy, approximately 1×107 cells were fixed with 1% formaldehyde and quenched by glycine. The cells were washed three times with PBS and then harvested in ChIP lysis buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl2, 0.2% Triton X-100). DNA was digested to 150-300 bp by MNase (SIGMA) before extensive centrifugation. Four volume of ChIP dilution buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) was added to the supernatant. The resulted lysate was then incubated with protein G beads and antibodies at 4ºC over night. The beads were washed 5 times and DNA was eluted by Chip elution buffer (0.1 M NaHCO3, 1% SDS, 20 µg/ml proteinase K). The elution was incubated at 65ºC over night and DNA was extracted with DNA purification kit (TIANGEN). RNA-Seq library construction was performed by using Illumina TruSeq kit, briefly, 10ug total RNA as initiation, and then mRNA was purification by oligo-dT magnetic beads. mRNA  was fragmented into 200-400bp and revers transcript into dscDNA for library construction. For ChIP-Seq library construction, cross-linked chromatin was fragmented into 150-300bp, and then fragmented DNA was used for library construction and sequencing by Illumina Hiseq2000.