anti-H3K27Ac Ab (Abcam, catalog# ab4729, lot# GR3216173-1)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq; Ten million of BM-derived DN3 cells with 1% formaldehyde for 10 min. The reaction was quenched by addition of 1/10 volume of 0.125M glycine and the cells were washed with HBSS (Gibco). Pelleted nucleus were dissolved in lysis buffer (0.5% SDS, 10 mM EDTA, 0.5 mM EGTA, 50 mM Tris-HCl (pH 8) and PIC) and sonicated on a Bioruptor (Diagenode) for 18 cycles of 30sec sonication followed by 30sec rest, with max power. Five ug per 10^7 cells of anti-H3K27Ac Abs (ab4729, Abcam) were hybridized to Dynabeads anti-Rabbit (Invitrogen) and added to the diluted chromatin complex. They were incubated over night at 4°C, then washed and eluted for 6hr at 65°C in ChIP elution buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA 50 mM NaCl, 1% SDS, and 50 ug/ml proteinase K). Precipitated chromatin fragments were cleaned up using Zymo ChIP DNA Clean & Concentrator. ChIP-seq libraries were constructed using NEBNext ChIP-Seq Library Preparation Kit (E6240, NEB) and sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt.