Chromatin Immunoprecipitation and Enrichment Analysis

The cells were fixed with 1% formaldehyde and quenched with 125 mM glycine. Then, the cells were centrifuged and resuspended in cell lysis buffer (150 mM NaCL, 50 mM Tris pH = 8, 1% Triton X-100, 1% nf-40, 0.01% sodium dodecyl sulfate (SDS), 1.2 mM EDTA pH = 8.0, 1 mM phenylmethylsulfonyl fluoride). The chromatin was broken by ultrasound, and reacted with HDAC3 or immunoglobulin G antibody (Millipore, Massachusetts, USA). Then, the cells were treated with RNase (Qiagen) and proteinase K (Roche, Basel, Switzerland) at 45 °C. DNA was eluted with 100 mM NaHCO₃ and 1% SDS, and reacted with 300 mM NaCl at 65 °C for 16 h. Qiaquick PCR purification kit (Qiagen) was adopted to purify the immunoprecipitated DNA and the extracted DNA. The purified DNA was amplified by Qiagen QuantiTech SYBR Green PCR master mix in RT-qPCR and subjected to enrichment analysis [21 (link)]. Each reaction was run in triplicate to obtain the average value.