GSM9290858: Medial preoptic area, 50 ug/kg BPA, Male, rep4; Rattus norvegicus; Bisulfite-Seq
DNA was extracted using the MagMAX FFPE DNA/RNA Ultra Kit (#A31881, ThermoFisher Scientific). For unfixed frozen tissue, 210 μl of protease solution was pipetted onto each MPOA sample and allowed to incubate at 55C at 900 rpm for at least one hour. The homogenate was immediately processed for RNA and DNA extraction using the Kingfisher Flex System following the manufacturer's instructions. Some DNA samples were extracted using the MagMAX DNA Multi-Sample Ultra 2.0 Kit following the manufacturer's instructions for tissue samples (#A45721, ThermoFisher Scientific). Library preparation for oxRRBS was done using the Ovation RRBS Methyl-Seq System with the TrueMethyl oxBS module (Tecan) using 55 ng of input DNA. Between 12-20 samples were processed per batch of library preparation with a total of 5 batches, balanced by prenatal treatment group and sex. The oxidation of DNA prior to bisulfite conversion converts 5hmC to 5fC and allows for discrimination between 5mC and 5hmC at single base-pair resolution. For this study, oxidative bisulfite conversion was used to remove 5hmC and detect only 5mC in the samples.