Methyl and hydroxymethyl DNA immunoprecipitation sequencing (MeDIP-seq and hMeDIP-seq)

The liver MeDIP-seq data was previously generated and reported in Yang et al.⁴⁷. The hMeDIP assay was performed using the MagMeDIP-seq Package (Diagenode) following the manufacturer’s protocol with a mouse antibody against 5hmC (Diagenode, C15200200-50); 1.2 µg of gDNA was sonicated into ~ 200 bp fragments using the S220 focused ultrasonicator (Covaris). To maximize IP yield for the liver hMeDIP-seq, samples were processed in duplicates then pooled before IPure purification. Prior to immunoprecipitation, samples were spiked with hydroxymethylated and unmethylated internal DNA controls. IP efficiency and success was verified by qPCR targeting internal DNA controls. The DNA amount was quantified by Qubit HS DNA kit (Thermo Fisher) and the fragment size was assessed on a 2100 BioAnalyzer using a DNA HS kit (Agilent). Individual libraries for immunoprecipitated DNA and 10% input were dual indexed (NEBNext Multiplex Oligos, unique dual indices, New England Biolabs), PCR amplified, and then pooled into equimolar amounts and further quantified using the NEBNext Library Quant Kit (New England Biolabs). The pooled library was subjected to 50 bp paired-end sequencing on the Illumina NextSeq 2000 platform using a P2 100-cycle kit (Illumina).