title: GSM8860043: Input_DB_ZHX2; Homo sapiens; ChIP-Seq
library_name: GSM8860043
library_construction_protocol: 5 million DB cells were fixed with 1% formaldehyde at room temperature for 10 minutes, quenched with 125 mM glycine and harvested. The harvested cells were sent to Active Motif China (Shanghai, China) for ChIP-Seq. Active Motif prepared chromatin, performed ChIP reactions, generated the libraries and performed basic data analysis. In brief, chromatin was isolated by adding lysis buffer and fragmented by sonication. DNA was sheared to an average length of 200–500bp with Active Motif's PIXUL® Multi-Sample Sonicator (Cat# 53130). Genomic DNA (Input) was prepared by de-crosslinking at 65°C for 4 hours followed by RNase A (Thermo Fisher) and Proteinase K (Thermo Fisher) digestion. The Input DNA was purified with QIAGEN PCR Purification Kit and quantified by Onedrop (Wins). Fragmented chromatin (for IP) was incubated with 15 μl of anti-ZHX2 (CST, Cat #43035) at 4℃ for overnight. 25 ul of rProtein A Magarose Beads (smart-lifesciences) were added to the samples and incubated at 4°C for 2 hours. Complexes were washed, eluted from the beads with SDS buffer, then de-crosslinked at 65°C for overnight. The de-crosslinked chromatin was subjected to RNase A and proteinase K treatment. ChIP DNA was purified with QIAGEN PCR Purification Kit and quantified with Qubit (Thermo Fisher). Illumina sequencing libraries were prepared from the ChIP-DNA and input DNAs with VAHTS Universal DNA Library Prep Kit for Illumina V4.