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

• Published in: Genome Biology Vol. 26; no. 1; pp. 244 - 13
• Main Authors: Chen, Xiufei, Cheng, Jingfei, Kong, Linzhen, Shu, Xiao, Xu, Haiqi, Inoue, Masato, Fernández-Berrocal, Marion Silvana, Døskeland, Dagny Sanden, Bjørås, Magnar, Sivakumar, Shivan, Liu, Yibin, Ye, Jing, Song, Chun-Xiao
• Format: Journal Article
• Language: English
• Published: England BioMed Central 18.08.2025; BMC
• Subjects: 5hmC; 5mC; Aging; Method; scCAPS; scTAPS; Single-cell sequencing; 5hmC; Neuron; scTAPS; Aging; Single-cell sequencing; 5mC; scCAPS
• ISSN: 1474-760X; 1474-7596; 1474-760X
• DOI: 10.1186/s13059-025-03708-1

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.