Genome-wide CpG Methylation Profiling via MeD-seq

MeD-seq assays were performed as previously described [20 (link)]. Briefly, 20 µL genomic DNA (input 90 ng) from frozen liver tissues was digested with LpnPI (New England Biolabs), generating 32 bp fragments around the methylated recognition site containing a CpG. These short DNA fragments were further processed using the ThruPlex DNA–seq 96D kit (Rubicon Genomics, Ann Arbor, MI, USA). Stem–loop adapters were blunt-end ligated to repaired input DNA, then amplified to include dual-indexed barcodes using a high-fidelity polymerase to generate an indexed Illumina NGS library. The amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA, USA). Multiplexed samples were sequenced on Illumina NextSeq2000 systems for paired-end reads of 50 bp, according to the manufacturer’s instructions. The dual-indexed samples were de-multiplexed using bcl2fastq v2.20 software (Illumina, San Diego, CA, USA).