GSM5769860: OB_H3K27ac; Homo sapiens; ChIP-Seq
Cells were harvested and cross-linked with 1% formaldehyde for 10 min and quenched with 0.125 M glycine for 5 min. Cells were lysed in 1 mL lysis buffer and rotated for 30 min at 4°C. Cell lysates were centrifuged at 2,400 g for 10 min at 4°C to isolate the nuclei. Nuclei were resuspended in digestion buffer and subjected to enzymatic digestion to shear chromatin fragments to an average size between 200 bp and 500 bp by incubating the tube at 37°C for 10-15 min.  The shearing reaction was stopped, and the fragmented chromatin was centrifuged at 18000 g for 10 min at 4°C. The supernatant was transferred to a new Eppendorf tube, and the ChIP reaction mix containing protein A/G magnetic beads, ChIP IP buffer, antibody, and protease inhibitor cocktail was added and rotated at 4°C overnight. After overnight incubation, protein A/G magnetic beads were washed. The chromatin was eluted in reverse cross-linking buffer followed by incubation of the tube at 65°C for 3 h. Then, the ChIP DNA was treated with RNase A and protease K at 37°C for 30 min and purified using phenol chloroform. ChIP DNA was processed for library generation using the QIAseq Ultralow Input Library Kit (QIAGEN) following the manufacturer's instructions.