Fig. 2

RRBS Project Workflow. Prior to methylation analysis, 6 pairs of fibroblasts isolated from the same animals at 5- and 16-months of age were selected and challenged with LPS for 0, 2, 8 and 36 h to measure cytokine protein and gene expression. In preparation for RRBS, fibroblast DNA was digested with MSPI to enrich for CpG rich regions of the genome, followed by end-repair and Poly-A tail addition. The digested DNA was then ligated to Illumina primers, size selected and bisulfite converted prior to sequencing. The subsequent reads were quality controlled by FastQC and Illumina adaptors were trimmed with TrimGalore. Those that passed quality control were then aligned to the BosTau8 genome and uniquely mapped reads were kept for analysis with a paired T test at each CpG site with greater than or equal to 5× coverage. Once differentially methylated sites were identified, clustering analysis was performed on the top 100 sites based on p-value. Functional analysis to identify enriched KEGG pathways, GO terms and transcription factors was performed by DAVID and gene expression analysis by RT-qPCR