First High Resolution Rat Methylome Figure 4. Average methylation density around transcription start site (TSS). A - Distribution of peak summit count in 100 bp sliding window, 5 kb upstream and downstream from the start site was calculated for all RefSeq genes and identified liver proteins. Count was normalized by dividing individual count with total number of genes in that category. The plot obtained of RefSeq and identified liver proteins were further smoothened by taking a moving average of 5. B – Similar distribution of peak summit count in 100 bp sliding window, 5 kb upstream and downstream from the transcription start site was calculated for up regulated and down regulated genes in normal rat liver tissue. Smoothing of peaks was done by taking moving average of 5. doi:10.1371/journal.pone.0031621.g004 data from the Alternative Splicing and Transcript Diversity (ASTD) database, selecting only the unique entries in intron retention (IR) class [32]. We found methylation levels within introns that are retained to be markedly higher compared to constitutive introns that are spliced out (Figure 8; Figure S9A). To confirm that these introns are indeed a part of the mRNA, we performed intron specific RT-PCR in rat liver tissue for a few such introns which were found to be methylated in our data. We isolated RNA from rat liver and converted it into cDNA by RT- PCR using primers that would specifically amplify the introns if Figure 5. Average methylation density at the intron-exon-intron junctions. Distribution of peak summit count in 10 bp sliding window, 200 bp upstream and downstream from the start site and end site of exons was calculated for all RefSeq gene exons, first exon and all last exons. Smoothing of peaks was done by taking moving average of 5. doi:10.1371/journal.pone.0031621.g005 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31621