Bisulfite sequencing

The CpG islands in the 5’-flanking region of RPRM gene (NCBI reference sequence: {"type":"entrez-nucleotide","attrs":{"text":"NC_000002.12","term_id":"568815596","term_text":"NC_000002.12"}}NC_000002.12) were predicted by MethPrimer [15] and we picked one pair of bisulfite sequencing primers (RPRM-F: 5’-GTTTTAGAAGAGTTTAGTTGTTG-3’; RPRM-R: 5’-CTACTATTAACCAAAAACAAAC-3’) which contains 30 C-G dinucleotides within the target sequence to study the RPRM gene methylation. The target reference sequences of human RPRM gene (Ref-seq), the corresponding bisulfite-converted sequences from fully-methylated (M-seq) or unmethylated (U-seq) genomic DNA were provided in S1 File.

For bisulfite sequencing, the PCR was conducted using the bisulfite primers and Ex Taq HS (TAKARA BIOTECHNOLOGY, DALIAN). The PCR products were purified using TaKaRa MiniBEST agarose gel DNA extraction kit (TAKARA BIOTECHNOLOGY, DALIAN) before integrated into the pGEM®-T Easy vector (Promega Biotech, Beijing). After transforming the E.coli DH5α competent cells, eight to ten clones were sequenced and then aligned with M-seq and U-seq for analysis of cytosine methylation using the BioEdit software [16].