Methylated DNA immune-precipitation sequencing (MeDIP-Seq) and hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-Seq)

Purified genomic DNA was sonicated to obtain fragments with a size range of 200–800 bp for hMeDIP-seq or 200–500 bp for MeDIP-seq. Following end repair of the DNA samples with the addition of deoxyadenosine and adaptor ligation based on the Illumina paired-end protocol, the DNA fragments were immunoprecipitated with anti-5-hydroxymethylcytosine or anti-5-methylcytosine antibody. Then, the precipitated fragments were amplified by PCR, and 300–900-bp DNA fragments were selected by using AMPure XP beads. The completed libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, captured on an Illumina flow cell, and amplified in situ. The libraries were then sequenced on the Illumina HiSeq 4000 following the HiSeq 3000/4000 SBS Kit (300 cycles) protocol. After base calling, low-quality reads were passed, and the clean reads were aligned to the UCSC human reference genome hg19 using HISAT2 software (V2.1.0).