Bisulfite treatment of genomic DNA was performed as previously described[33 (link)], using glycogen as carrier, and the precipitated DNA was redissolved in TE buffer, amplified by PCR and sequenced directly. The primers were designed to cover 16 CpG sites in the promoter region in NDRG2 (Figure 1A) and their sequences were (5’-3’): TTTTCGAGGGGTATAAGGAGAGTTTATTTT and CCAAAAACTCTAACTCCTAAATAAACA[34 (link)]. A positive control with in vitro methylated (IVM) DNA was prepared by mixing 2 μL NEB2 buffer, 1 μL 20 x S-adenosylmethionine (New England Biolabs, B9003S), 200 ng reference human genomic DNA and 1 µL SssI methyltransferase (New England BioLabs, M0226S) in a total of 20 μL. Samples were incubated at 37 °C overnight with occasional addition of 2 μL 20 x S-adenosylmethionine to ensure sufficient methyl-donor substrate. The following description was used for each CpG site: Unmethylated (no methylation signal); weakly methylated (methylation signal was less than or approximately equal to unmethylated signal); and strongly methylated (methylation signal was greater than unmethylated signal).