title: GSM7785468: PMA_J_input; Homo sapiens; ChIP-Seq
library_construction_protocol: In brief, chromatin was isolated by adding lysis buffer and fragmented by sonication. DNA was sheared to an average length of 200-500 bp with EpiShear probe sonicator (Active Motif, 53051). The same amount of Drosophila chromatin (Active Motif, 53083) was incubated with sheared chromatins from THP-1 cells and PMA-treated PMA cells, respectively for ChIP normalization. Genomic DNA (Input) was prepared by de-crosslinking at 65°C for 4 hours followed by RNase A and Proteinase K digestion. The Input DNA was purified with PCR Purification Kit (Qiagen, 28004) and quantified (Onedrop, Wins).Fragmented chromatin mix (for IP) was incubated with 5 μl of anti-C/EBPα (Cell Signaling Technology, 8178) and 1 μg of Drosophila H2A.v antibody (Active Motif, 61686) at 4°C for overnight. 25 μl of rProtein G Magarose Beads (Smart-Lifesciences, SM004005) 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 PCR Purification Kit (Qiagen, 28004) and quantified (Thermo Fisher, Qubit). Illumina sequencing libraries were prepared from the ChIP-DNA and input DNAs with VAHTS Universal DNA Library Prep Kit for Illumina V4.