Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was evaluated as described previously (Subbanna et al., 2014, Subbanna et al., 2015, Subbanna et al., 2018). Briefly, adult mouse hippocampus (HP) tissue (25 mg) was fixed with formaldehyde (1%), homogenized, and subjected to DNA shearing. The sample amount was normalized to contain equivalent protein amounts. A small aliquot of precleaned chromatin was used as the input. Chromatin immunoprecipitation was performed with an antibody recognizing normal rabbit IgG as a negative control and with anti-rabbit-H3K14ac (monoclonal, # 7627), anti-rabbit-H4K8ac (polyclonal, # 2594), anti-rabbit-G9a (monoclonal, # 3306), anti-rabbit-H3K9me2 (monoclonal, # 4658) (Cell Signaling Technology, Denvers, MA USA), and anti-rabbit-CBP (polyclonal, # ab2832, Abcam, Cambridge, MA, USA) antibodies. The real-time qPCR with mouse primers (forward, 5’-AGTGCTCTGGCGAGTAGTCC-3’ and reverse, 5’-TCGGGACAGGCTAAGAACTC-3’) designed to amplify Arc gene promoter regions was followed to quantify the immunoprecipitated DNA and data were normalized to the total DNA (input). Relative enrichment of H3K14ac, H4K8ac, CBP, H3K9me2 and G9a on the Arc gene promoter in the saline and treatment groups was determined by taking the ratio between immunoprecipitated chromatin and input chromatin (Hendrickx et al., 2014). Data presented as % input.