GSM4743061: NPC H3K27me3; Homo sapiens; ChIP-Seq
Genomic DNA was extracted by PureLink™ Genomic DNA Kit according to manufacturer's protocol. Total RNA of each cell type was extracted by TRIzol™ Reagent (Invitrogen) and RNA integrity (RIN) was assessed by Agilent Bioanalyzer (RIN>=8). 1 million cells were crosslinked in crosslinking buffer (100 mM NaCl, 0.1mM EDTA, 5mM Hepes, pH 8.0, 1% formaldehyde) for 10 min at 25°C. The crosslinking was quenched with 125 mM glycine in 25°C for 5 min with rotation and washed twice with ice-cold PBS. The cross-linked cells were re-suspended in SDS lysis buffer (1% SDS, 50 mM Tris-HCl, pH8.0, 10mM EDTA) with protease inhibitor (Roche). Mono- and di-nucleosome size chromatin was obtained through sonication (Covaris, S220) Total 300ng of genomic DNA was used for bisulfite conversion (EZ DNA Methylation-Gold Kit, Zymo research) and library preparation (TruSeq DNA Methylation, Illumina). We sequenced the bisulfite converted genomic library (2x150bp) using HiseqX. 200ng of total RNA was used for library preparation with TruSeq rapid SBS kit or Truseq SBS Kit v4 according to the kit guidelines. We sequenced the paired-end RNA-sequencing library (2x101bp) using HiSeq 2500 system. The immunoprecipitated DNA was recovered using AMPure XP beads (Beckman Coulter) and ChIP-seq libraries were prepared using NEBNext Ultra II DNA library Prep Kit following the manufacturer's instruction. The ChIP-seq libraries were sequenced in 150 bp paired-end mode using Illumina NovaSeq 6000 platform.