Figure 1

Workflow of the RRHP assay. Genomic DNA was first digested by restriction endonuclease MspI and then ligated with the modified adapters such that the MspI site is reconstituted at the junction of P5 adapter and fragment but not at the junction of P7 and fragment. The adapter-ligated fragments are either homo- or hetero-adapterized. Homo-adapterized fragments have the same adapters at both ends (P5-P5 or P7-P7), which will form an inhibitory stem-loop hairpin preventing itself from further amplification, whereas the hetero-adapterized fragments have different adapters (P5-P7). All ligated library fragments are glucosylated with β-glucosyltransferase (β-GT). Fragments presenting 5hmC at the junction and, thus glucosylated, are resistant to a second MspI digestion while fragments with unmodified C or 5mC are cleaved, removing the P5 adapter. Only the fragments with both P5 and P7 adapters after the second MspI digestion will be amplified and sequenced.