Bisulfite conversion was carried out using EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Nested polymerase chain reaction amplifications were performed with a standard polymerase chain reaction protocol in 25-ml volume reactions containing 3–4 μl of sodium-bisulfite-treated DNA, 0.2 μm primers and master mix containing Taq DNA polymerase (Sigma-Aldrich, St Louis, MO, USA). Primer sequences can be found in Supplementary Table 2. Polymerase chain reaction amplicons were processed for pyrosequencing analysis according to the manufacturer’s standard protocol (Qiagen, Gaithersburg, MD, USA) using a PyroMark MD system (Qiagen) with Pyro Q-CpG 1.0.9 software (Qiagen) for CpG methylation quantification.
