Epityper DNA methylation anlaysis is based on bisulfite conversion of DNA, PCR amplification, followed by in vitro transcription and analysis of cleaved products by Mass Spectrometry (MALDI-TOF). The primers were designed using MethPrimer (www.urogene.org/methprimer) for 33 gene promoter regions with 48 amplicons (103–625 bp with median target length of 400 bp) covering on average of 9 CpGs per amplicon (Suppl. S5). In brief, DNA was isolated as described above for MeDIP analysis from a single embryo with or without alcohol treatment (n = 3 each). The methylation detection was carried in Sequenom facility at San Diego, CA. Approximately 1 µg of DNA from each sample was treated with sodium bisulfite using the EZ DNA methylation kit (ZymoResearch, CA) according to manufacturer’s protocol, and amplification was done using the primers with the T7 promoter tag on the reverse primer. The in vitro transcription was done, followed by site-specific cleavage using MassCLEAVE biochemistry. MassARRAY compact MALDI-TOF mass was used to acquire spectral peaks and converted into methylation ratios using EpiTYPER software.66 Two statistical criteria were used to identify genes validated by sequenom;
