The goal of the study was to determine how ethanol affects both DNA methylation and gene expression in a coordinated mechanism. Gene expression studies began with total RNA from triplicate biological samples. Each sample was biotinylated. cRNA was labeled, purified and fragmented, and hybridized with the Affymetrix Human Genome U133 plus 2.0 Array. Genomic DNA was isolated and the percentage of 5mC marking was quantified using an ELISA-like technique. For methylation mapping, total genomic DNA was isolated and sent to a service lab (Zymo Research) for processing. DNA was treated with bisulfite to convert unmethylated C residues to U and then the DNA was fragmented for standard whole-genome sequencing. While details of bioinformatic sequence analysis were not provided, presumably an algorithm similar to Bismark (Krueger, Andrews 2011) was used to align bisulfite-transformed sequences and count methylated nucleotides. A typical approach for this is to align with two separate C→U or G→A substituted genome sequences, one to model methylation for each of the two strands. Results showed a global increase in methylation, particularly near transcription start sites (TSS) and CpG islands,
