Chromatin Immunoprecipitation for Epigenetic Analysis

Chromatin immunoprecipitation was performed as previously described using the Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions [36 (link),55 (link),56 (link),57 (link)]. Sheared chromatin (90 μL) from the prefrontal cortex or hippocampus was used for immunoprecipitation and incubated at 4 °C overnight with primary antibodies (anti-H4R3me2a, anti-H3K4me3, antiH3K36me3, and anti-MeCP2; Abcam, Cambridge, UK) or normal rabbit IgG (negative control, supplied in the kit). Meanwhile, 5 μL of sheared chromatin was saved as input for normalization. Follow-up purified gDNA from each antibody was used at the BDNF exon IV, MBP, and SOX10 promoter region via real-time PCR (Table 2). The percentage input was calculated using the following formula: ΔCt [normalized ChIP] = (Ct [ChIP] − (Ct [Input] − Log2 (Input dilution factor), where dilution factor = 5/90 = 18.5. Finally, input%  =  100/2^{ΔCt [normalized ChIP]}, with this value delegating the enrichment of epigenetic modifications in specific regions.