GSM5005292: HeLa_WT_H3K18ub_ChIP_seq_rep; Homo sapiens; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 5 minutes. After quenching with 125 mM glycine, the cells were harvested and digested by MNase (NEB, M0247S, 2 U/μL) at 37 °C for 20 minutes to produce soluble chromatin with DNA fragments in the range of 150-300 bp. The lysates were then sonicated for 5 minutes (30 sec on / 30 sec off) using Bioruptor Sonicator (Diagenode, Inc., Denville, NJ) and centrifuged at 21,130 g for 10 minutes. The supernatants were collected and the chromatin content was incubated with Protein A and Protein G Dynabeads pre-linked with antibodies against H3K27me3 or H3K18ub overnight at 4 °C. Bound chromatin was washed extensively, then eluted and reverse-crosslinked at 65 °C overnight using elution buffer. After being treated by RNase A and proteinase K, the samples were extracted by phenol: chloroform and precipitated with ethanol in the presence of linear acrylamide. After dissolving in nuclease free TE buffer, the samples are ready for sequencing library construction. ChIP-seq libraries were prepared for sequencing using standard Illumina protocols.