RRBS-seq quantification and differential methylation analysis

Raw datasets were downloaded from the GEO repository under accession ID {"type":"entrez-geo","attrs":{"text":"GSE80672","term_id":"80672"}}GSE80672. Raw sequence reads were trimmed using Trim Galore! (v0.4.2). Trimmed sequences were aligned using Bismark [151] (v0.16.3). Data visualization and analysis were performed using SeqMonk and custom RStudio scripts. Data from replicates of the same treatment group were merged using SeqMonk’s data group option, in order to enhance coverage and detection of subtle differences. To reduce the effect of coverage differences between samples, only cytosines covered by at least five observations were used for the differential methylation analysis. Differentially methylated cytosines (dCpGs) were identified by pairwise Chi-squared tests with subsequent multiple testing correction (adjusted p-value <0.05) and a minimal difference cutoff of 10%. Filtering of CpGs and binning was conducted for old DR treated C57BL/6♂ or B6D2F1♂ mice including controls (S1 Fig).