2.3. Sodium bisulfite pyrosequencing

Bisulfite conversion was carried out using EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. Nested PCR amplifications were performed with a standard PCR protocol in 25 ul volume reactions containing 3–4 μl of sodium-bisulfite-treated DNA, 0.2 uM primers, and master mix containing Taq DNA polymerase (Sigma Aldrich, St. Louis, MO). Primer sequences can be found in Table S1. PCR amplicons were processed for pyrosequencing analysis according to the manufacturer’s standard protocol (QIAGEN, Germantown, MD) using a PyroMark MD system (QIAGEN) with Pyro Q-CpG 1.0.9 software (QIAGEN) for CpG methylation quantification. Sample processing order was randomized by sorting on a random number generated in Perl. Laboratory personnel were blind to mood status until the completion of data generation.