Single-Cell 5-Formylcytosine Landscapes of Mammalian Early Embryos and ESCs at Single-Base Resolution

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present “CLEVER-seq” (chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells. [Display omitted] •A bisulfite-free method detects 5fC at the single-cell and single-base resolution•5fC is highly dynamic and enriched in functionally important genomic regions•Intrinsic 5fC heterogeneity is revealed for mouse early embryos, mEpiSC, and mESCs•Emergence of promoter 5fC production precedes the upregulation of gene expression CLEVER-seq is a single-cell, single-base resolution whole-genome 5fC-sequencing technology. CLEVER-seq reveals intrinsic 5fC heterogeneity in mouse early embryos, EpiSCs, and ESCs and dissects its highly patterned genomic distributions. During mouse pre-implantation development, 5fC shows parental-specific signatures, and promoter 5fC production precedes the upregulation of corresponding gene expression.