Bisulfite Conversion and Sequencing

Genomic DNA from AVP and OT PVN punches (whole sample) and cultured cells (200 ng) was bisulfite converted using Zymo EZ DNA Methylation-Gold kit (Cambridge Bioscience) as previously described (Greenwood et al., 2016a (link)). Primers for amplification of bisulfite converted DNA were designed using MethPrimer software; (mouse 5′-TTAGAGAGTTGAGTTAGTTAAGGAAA-3′ and 5′-CAAAAATCTCTCTAAATCTCTTCC-3′; rat 5′-AGGAAGTTTATAGTTTTTAGGATAG-3′ and 5′-AAAAATCTCTCTAAATCTCTTCCTC-3′; human 5′-TTGGAAGGATGGAAATAGTTTT-3′ and 5′-AAACCCCTAACTAACTAACCCAACTA-3′). The converted DNA was amplified using Platinum Taq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with the following cycling parameters: 94°C for 2 min followed by 45 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 2 min. The PCR products were purified using Qiagen’s PCR purification kit and ligated into pGEM-T Easy vector (Promega, Madison, WI, USA). Ten independent clones were sequenced per sample.