GSM1643802: Sample9_NPC_p53_4F-H3K4me3; Homo sapiens; ChIP-Seq
3*105 cells were fixed using 1% PFA, treated with glycine 0.1M and resuspended in nuclei lysis buffer (SDS 1%, EDTA 100mM, Tris-HCl 50mM pH7.5). Chromatin samples were sonicated with a Branson Digital Sonifier to generate DNA fragments from 200 to 1000 bp, diluted with ChIP RIPA Buffer (Tris-HCl 0.1mM pH7.5, EDTA 1mM, EGTA 0.5mM, Triton X-100 1%, SDS 0.1%, Deoxycolic acid 0.1%, NaCl 140mM) and incubated with Dynabeads Protein A (Invitrogen) coupled to the specific antibody. After overnight incubation, the immunocomplexes were eluted in ChIP elution buffer (Tris-HCl 20mM pH7.5, EDTA 5mM, NaCl 140mM, SDS 1%, proteinase K 40mg/ml) and the crosslinking was reverted overnight at 65ºC. DNA was recovered by phenol/chloroform extraction and quantified by quantitative PCR using SYBR green on an ABI Prism 7300 system. ChIP DNA was prepared into ChIP-Seq libraries using NEBNext Ultra DNA Library Prep Kit for Illumina according to manufacturer's instructions except adapters were purchased from Bioo Scientific.  Briefly, ChIP DNA was end-repaired, adenylated and adapter ligated. Adapter-ligated DNA was subjected to 12 cycles of PCR using NEB Q5 PCR mastermix. AMPure beads were used to purify sequencing libraries from reactions (Beckman Coulter). ChIP-Seq libraries were quantified using Invitrogen Qubit, Agilent Tape station and Kapa library quantification qPCR kit. Libraries were pooled and sequenced single-end 50bp on Illumina HiSeq 2500.