GSM3598038: [ChIP-seq, H3K27ac] MEF rtTA input (rep2); Mus musculus; ChIP-Seq
Approximately 30 million cells were used for each experiment. Chromatin was sheared using the Covaris sonicator until DNA was fragmented to 200-500bp. For each reaction 1% of sonicated chromatin was kept as input DNA. 5ug of H3K27ac (Abcam ab4729) antibody was added to remaining chromatin and incubated overnight at 4°C. 100uL of protein G Dynabeads (Thermo Fisher; 10003D) were added to each ChIP reaction and incubated for at least 4h at 4°C. Beads were then washed, eluted and reverse cross-linked overnight. After reverse cross-linking, isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water. ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier's protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.
GEO Accession
GSM3598038