mRRBS was performed as previously reported (45). Briefly, genomic DNA was digested with Msp I (New England BioLabs) before size selection of 100- to 250-bp fragments with solid-phase reversible immobilization beads (MagBio Genomics). DNA was bisulfite converted with the EZ DNA Methylation-Lightning Kit (Zymo Research). Libraries were prepared with the Pico Methyl-Seq Library Prep Kit (Zymo Research) using Illumina TruSeq indices and sequenced using single-end reads (NextSeq 500, Illumina) with a 500/550 V2 High Output reagent kit (1 × 75 cycles).

Bioinformatic processing and alignment of the sequenced libraries to the hg19 reference genome were performed as previously reported (45). Well-observed CpG positions were obtained by performing an analysis of variance (ANOVA)–like test for differential methylation with the DSS v2.26.0 R/Bioconductor package (46) and quantified using the SeqMonk platform (v1.40.1) with the bisulfite feature methylation pipeline. Metagene-style plots of all well-observed CpGs were generated in SeqMonk as a quantitation trend plot and visualized with GraphPad Prism v7.04.

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