The probe-level analysis was extended to the entire gene-related promoter and CpG island regions covered by the NimbleGen array, from 1,300-bp upstream to 500-bp downstream of the transcription start site, which ensured characterization of methylation patterns in distal regulatory regions without diluting the signals in the core promoter with increased noise levels. Since sheared DNA fragments ranged from 200–1,000 bp, and each probe covers approximately 50 bp, our conservative analysis used two consecutive probes on the NimbleGen CpG island array (covering 150–200 bp). Using a linear mixed model, we identified DNA regions with increased/decreased methylation levels based on the signal intensities of two consecutive probes between control and alcohol-treated samples. Depending on the NimbleGen probe selection, each promoter or CpG island contains more than one consecutive probe pair. By average, there are 17 probe pairs for each gene. We identified genes that contained no less than one DNA region (two consecutive probes) with increased/decreased methylation levels within 1,300-bp upstream and 500-bp downstream of transcription start sites.
