MeD-seq: Profiling Methylated DNA Fragments

MeD-seq analyses were essentially carried out as previously described [22 (link)]. In brief, 22 DNA samples were digested by LpnPI (New England Biolabs, Ipswich, MA, USA), resulting in snippets of 32 bp around a fully methylated recognition site that contains 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 repair input DNA and 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 HiSeq2500 systems for single read of 50 bp according to manufacturer’s instructions. Dual indexed samples were de-multiplexed using bcl2fastq software (Illumina, San Diego, CA, USA).