Total genomic DNA was isolated and 200 ng of DNA was subjected to methylated DNA quantification assay by using MethylFlash methylated DNA quantification kit (Epigentek Inc., Brooklyn, NY). % 5-methyCytosine was quantified against a positive standard provided. Alternatively, total genomic DNA was isolated and processed for genome-wide DNA methylation sequencing analysis by Zymo Research (Irvine, CA). Briefly, DNA samples were fragmented and end-modified to add an adaptor. After size selection, bisulfite conversion was performed and amplification was done for library construction. Next-generation bisulfite sequencing was done to obtain raw data. After sequence quality check, bioinformatics processing was performed.
