title: GSM6369197: ChIP-Seq_HEK293T_HDAC2; Homo sapiens; ChIP-Seq
library_construction_protocol: For ChIP assays, cells were fixed with disuccinimidyl glutarate (DSG) (2 mM) (Proteochem) for 45 mins at RT, washed twice with PBS and then double-fixed with 1% formaldehyde for another 15 mins at RT (HA-tagged proteins (Abcam, ab9110), CHD4 (Abcam, ab70469), HDAC2 (Santa Cruz Biotechnology,  sc7899), GATAD2A (Abcam, ab87663), and KDM1A (Bethyl Laboratory, A300-215A). Fixation was stopped by adding glycine (0.125 M) and incubating for 5 mins at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 300-500 bp average in size through sonication. Resultant was immunoprecipitated with control IgG or specific antibodies overnight at 4 ℃, followed by incubation with protein G magnetic beads (Biorad) for an additional 2 hrs. After washing and elution, the cross-linked protein-DNA complex was reversed by heating at 65 ℃ overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to either qPCR analysis or high throughput sequencing. The libraries were constructed following Illumina ChIP-seq Sample prep kit. Briefly, ChIP DNA was end-blunted and added with an 'A' base so the adaptors from Illumina with a 'T' can ligate on the ends. Then 200-400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.