title: GSM8059866: Zbtb21K419/845R double mutant; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were lysed in 1 mL lysis buffer, and rotated for 30 min at 4℃. Cell lysates were centrifuged at 2,400 x g for 10 min at 4℃ to isolate the nuclei. 1.Nuclei were suspended in digestion buffer and subjected for Enzymatic shearing to shear chromatin fragments to an average size between 200 bp and 500 bp by incubate the tube at 37℃ for 10-15 min.  2.Stop the shearing reaction and the fragmented chromatin was centrifuged at 18000 x g for 10 min at 4℃.  3.Supernatant was transferred to a new Eppendorf tube and added the chip reaction mix which containing proteinA/G magnetic beads, chip IP buffer, antibody, protease inhibitor cocktail, and rotated at 4℃ overnight. 4.After overnight incubation, proteinA/G magnetic beads were washed. The chromatin was eluted in reverse cross-linking buffer followed by incubate the tube at 65℃ for 3h. Then the ChIP DNA was treated with RNase A and proteinase K at 37℃ for 30 min, and purified using Phenol chloroform. 5.ChIP DNA was processed for library generation using the QIAseq Ultralow Input Library Kit (QIAGEN) following the manufacturer's protocol.