GSM5763698: MGT_H3K27ac_rep1; Mus musculus; ChIP-Seq
Chromatin were harvested using truChIP® Chromatin Shearing Kit (Covaris, PN 520154) following the manufacturer's protocol. Briefly, five million cells were cross-linked with Fixing Buffer A at room temperature for 10 minutes. Quenching Buffer E was added to the fixed cells and the cells were incubated at room temperature for an additional 5 minutes. The cells were washed twice with ice-cold PBS. To extract the nuclei, Lysis Buffer B containing 1x protease inhibitors were added to the cells. Incubate the cell on ice for 10 minutes on a rocker at 4 degree. Intact nuclei were collected by centrifugation at 1,700 x g for 5 minutes at 4 degree. The nuclei were washed once with Wash Buffer C and then Shearing Buffer D3.   The pelleted nuclei were resuspended in 1ml Shearing Buffer D3 and transfer to  milliTUBE (Covaris, PN 520135). The chromatin shearing were performed with E220 Focused-ultrasonicator (Covaris) with 60sec on and 30sec off, 14 cycles. The chromatin solution was clarified by centrifugation at 20,000 g at 4C for 45 minutes and then pre-cleared with Dynabeads protein A (Life Technologies) for 2 hours at 4C. The pre-cleared chromatin sample was incubated with 50 mL of Dynabeads protein A loaded with 5 mg antibody (anti-H3K27ac, ab4729; anti-Mef2c, ab211493; anti-Ascl1, ab74065) overnight at 4C. The beads were washed three times with 0.1% SDS lysis buffer, once with 0.1% SDS lysis buffer/0.35 M NaCl, once with 10 mM Tris-Cl (pH 8)/1 mM EDTA/0.5% NP40/ 0.25% LiCl/0.5% NaDOC, and once with TE buffer (pH8.0). The immunoprecipitated material was eluted from the beads by heating for 45 minutes at 68C in 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS. To reverse the crosslinks, samples were incubated with 1.5 mg/ml Pronase at 42C for 2 hr followed by 67C for 6 hr. The samples were then extracted with MinElute PCR purification Kit (QIAGEN) and eluted in TE buffer. ChIP DNA were then sent to UNC Chapel Hill's High-Throughput Sequencing Facility libraries for ChIP-seq library preparation with Thruplex DNA Seq Library Prep kit (Takara).