Both average-level and probe-level analyses were performed. In the average-level analysis, we focused on the regulatory regions near transcription start sites (from 1,300-bp upstream and 500-bp downstream of the transcription start sites). This approach avoided noise caused by distal oligonucleotides residing in the upstream intergenic regions and provided a more focused analysis of the region where most transcription factors bind, while allowing our results to be compared with other studies focused on human promoters.54 To closely evaluate methylation patterns in relationship to GC content within regions showing methylation changes, we further examined the GC content of differentially methylated promoter regions at the two-consecutive probe level (the region that encompasses two differentially methylated consecutive probes and their immediate upstream and downstream regions not covered by any other probe, ~250-bp). Regions with over 50% GC content and 0.6 CpG observed to expected ratio were considered as high-CpG regions and others were considered as low-CpG regions.
