GSM3583629: P4936 MYC MS2 24HR TC1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
Sequenced DNA Library
Chromatin immunoprecipitation sequencing. ChIP-seq for c-Myc, Max, and H3K27ac was performed as in (Lin et al., 2012) with minor changes. P493-6 cells were grown to a density of 1 × 10^6 cells / mL prior to the start of time course. At each time point, pellets of 50 million cells were isolated and cross-linked with 1% formaldehyde (10 min) followed by quenching (125 mM glycine). Cells were washed in cold PBS and harvested by cell scraper in cold PBS with protease inhibitors (Roche). Cells were centrifuged at 1,650g for 5 min and flash frozen and stored at –80°C at 5 × 106 cells per pellet. Pellets were resuspended in cytosolic and then nuclear lysis buffer, and DNA was sheared at 4 °C using a water-bath sonicator (Bioruptor, Diagenode) for 25 min at high output (30 s on, 30 s off) in 1 ml of sonication buffer supplemented with 0.5% SDS. Sonicated lysates were cleared by centrifuging at 20,000g for 10 min and incubated overnight end over end at 4 °C with magnetic beads prebound with antibody. Beads were washed three times with sonication buffer, one time with sonication buffer with 500 mM NaCl added, once with LiCl wash buffer (20 mM Tris pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE. DNA was eluted in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS). Cross-links were reversed overnight at 65 °C. RNA and protein were digested with 0.2 mg/ml RNase A for 2 h followed by 0.2 mg/ml proteinase K for 1 hour. DNA was purified with phenol chloroform extraction and ethanol precipitation. Libraries for sequencing were prepared using the Rubicon Thruplex DNA-seq/FD library preparation kit. An input of 50 ng of DNA or less was used, and following ligation, libraries were amplified per the manufacturer's instructions. Amplified libraries were then size selected using AMPure beads (Agencourt AMPure XP) according to the manufacturer's instructions. Further size selection was performed using a 2% gel cassette in the Pippin Prep (SAGE Sciences) set to capture fragments of 200–700 bp in size. Libraries were multiplexed at equimolar ratios and run on a NextSeq 500 instrument (75-bp, single-end reads).