Single-cell 5-formylcytosine Landscapes of Mammalian Early Embryos at Single-base Resolution

Active DNA demethylation in mammals involves Ten-eleven translocation (TET) family proteins-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC, an oxidized derivative of 5mC) at the single-cell level remains unexplored. Here we present “CLEVER-seq”, 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 of mouse early embryos reveals the highly patterned genomic distribution and parental specific dynamics of 5fC during mouse early pre-implantation development and synergistic 5fC production in paternal and maternal genomes. 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 early mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.