ChIP-seq Analysis of H3K9me2 in Arteriosclerosis

ChIP was performed as described in the Methods in the online-only Data Supplement. For genome-wide analysis, the DNA SMART ChIP-seq kit (Clontech, 634865) was used to generate Illumina-compatible sequencing libraries from 100 pg to 2 ng of DNA from 2 independent H3K9me2 ChIP experiments and associated input. Libraries were sequenced (Illumina NextSeq) using paired-end 75 bp reads. ChIP-seq reads were trimmed using Cutadapt v1.9,³⁷ aligned to the mouse GRCm38 genome using Bowtie2 v2.2,^{38 (link)} and reads per gene promoter (within ±1 kb of the transcription start sites) quantified using SeqMonk v1.4 (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk). Genes showing fewer than 20 read counts in the input samples were removed from further analysis and the ratio of H3K9me2/input signal was computed. Of genes with H3K9me2/input ratios in the top 25th percentile, 63 genes were associated with arteriosclerosis according to the Cardiovascular Disease Portal^{39 (link)} (https://rgd.mcw.edu/rgdCuration/?module=portal&func=show&name=cardio).