Direct and bisulfite-free 5-methylcytosine and 5-hydroxymethylcytosine sequencing at single-cell resolution with scTAPS and scCAPS + .

We present direct sequencing methodologies, scTAPS for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) and scCAPS + specifically for 5hmC, enabling quantitative detection of 5mC and 5hmC at single-base resolution and single-cell level. Achieving approximately 90% mapping efficiency, our plate-based methods accurately recover 5mC and 5hmC profiles in CD8 + T and mouse embryonic stem cells. Notably, scCAPS + reveals a global increase in 5hmC across neuronal and non-neuronal cells in the hippocampus of aging mice. Our methods offer strong potential for seamless integration into high-throughput single-cell multi-omics, facilitating future investigations of epigenomic dynamics in specific biological processes.