Direct, bisulfite-free 5mC and 5hmC sequencing at single-cell resolution
We report the first direct sequencing methodologies, termed scTAPS and scCAPS+ (exclusive to 5hmC), which enable quantitative detection of 5mC and 5hmC at single-base resolution and single-cell level. With ~90% mapping efficacy, our methods accurately benchmark 5mC and 5hmC profiles in CD8+ T and mES cells, respectively. Notably, scCAPS+ revealed a global increase in 5hmC within the hippocampus of aging mice, particularly evident in non-neurons. Overall design: scTAPS and scCAPS+ were benchmarked in human CD8+ T cells and mouse embryonic stem cells (mESCs) respectively to validate their performance. Furthermore, we utilized scCAPS+ to elucidate the dynamics of aging-related 5hmC in the hippocampal region, examining both neurons and non-neuronal cells derived from young (3 months) or aged (18 months) mice.