To analyze genome wide DNA methylation of NSC cultures following ethanol treatment, CpG methylation was assayed using an Affymetrix Mouse Promoter-specific microarray probed with bisulfite-treated DNA (Hicks et al. 2010). This microarray includes probes for regions within 8-10 kilobase pairs (kb) of the start codon for all RefSeq genes, with roughly three fourths of all CpG islands covered by more than 10 probes. Probe signals exceeding that of controls (FGF2 treated cultures), and therefore rich in methylated sequences, were associated with the neighboring gene and filtered for being within 30 kb of a CpG island. Parameters for comparisons between methylation and gene expression required at least a 3-fold increase in methylation signatures and at least a 2-fold decrease in transcript expression. 83 genes in total were both downregulated via microarray analysis and hypermethylated. The major gene clusters derived from this list included DNA metabolic and replication genes with 20% of all genes in these categories being hypermethylated.
