MeDIP-seq Library Preparation Protocol

cfDNA was used for the preparation of the MeDIP-seq library with some modifications [27 (link)]. Briefly, we used the Illumina NEBNext Ultra II DNA Library Preparation Kit (NEB, E7645) and ligated ~ 50 ng of cfDNA to the Illumina adapter according to the manufacturer's instructions. The resulting library was denatured at 95 °C for 10 min, immediately incubated on ice for 10 min, and then immunoprecipitated with 5-methylcytosine (5-mC) monoclonal antibody (Epigentek, A-1014). The MeDIP DNA was amplified with Q5 high-fidelity DNA polymerase (NEB, M0491), and the amplified products were purified with AMPure XP beads (Beckman). The amplified libraries were evaluated using a Bioanalyzer 2100 system (Agilent Technologies), and deep sequencing was performed using an Illumina HiSeq 2000 system.