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In vitro validation of BIND&MODIFY resolution with single base 5mC containing lambda DNA. A fragment of 700 bp lambda DNA was amplified by PCR, and 5mC was introduced near the end of forward strand only by the modified primers with the precise 5mC site. The 5mC labeled DNA was bound by 5mC antibody and was subsequently treated by BIND&MODIFY method. The m 6 A probability (Megalodon calling probability) of forward strand was calculated and plotted with loci. When the methylation probability cut-off was set at 0.53, the high methylation probability region was observed in 3′ end, overlapping with the expected the 5mC site

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