Single-Cell 5-Hydroxymethylcytosine Landscapes of Mammalian Early Embryos at Single-Base Resolution

DNA methylation and hydroxymethylation are extensively reprogrammed in mammalian early embryogenesis. However, a comprehensive picture of DNA hydroxymethylation map across early embryo stages is lacking. Here, we develop “schmC-CATCH” (single-cell 5hmC chemical-assisted C-to-T conversion-enabled sequencing) to obtain quantitative, genome-wide landscape of the base-resolution DNA hydroxymethylome in mouse gametes and pre-implantation embryos from one-cell (PN1-PN5) to blastocyst stages. We revealed the dynamics of DNA hydroxymethylation and observed dramatically different 5hmC patterns on the paternal and maternal genomes. We found hotspots of DNA hydroxymethylation during mouse early embryo development, which are highly associated with young retroelements including LINE1. 5hmC is also associated with regulatory elements, indicating a potential epigenetic function during early embryogenesis. In addition, 5hmC is asymmetrically distributed at the chromosome level and can be used to track the cell and strand lineages during the first two embryo cleavages. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for functional studies of epigenetic reprogramming in single cells. Overall design: We sought to address challenges in dissecting single-cell 5hmC patterns using a quantitative and genome-wide profiling method. We developed schmC-CATCH, an assay combining bisulfite-free chemical labelling with improved library construction for high sensitivity and precision.We adapted our previous chemical strategy to reproducibly label 5hmC in single cells for subtraction-free 5hmC readout, which avoid unacceptable noise signal and templates degradation. We collected gametes and early embryos including zygote, 2-cell, 4-cell, 8-cell, 16-cell and blastocytes by crossing C57BL/6J female mice with the DBA/2N male mice. All samples were extensively washed or purified to remove any somatic, gametic or polar body contaminants. In total, we performed 5hmC analysis of 456 individual cells from sperms, MII oocytes and preimplantation embryos.