Genome-wide stability of DNA replication program in single mammalian cells

We report the establishment of a single-cell DNA replication sequencing method, scRepli-seq, which is a simple genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct replication profiles, which were conserved from cell to cell. Haplotype-resolved scRepli-seq revealed similar replication timing profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication timing heterogeneity was present, and we discovered that developmentally regulated domains are a source of such variability, suggesting a link between cell-to-cell heterogeneity and developmental plasticity. Together, our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into 3D genome organization. Overall design: BrdUIP Repli-seq, Population Repli-seq, 100-cells Repli-seq and single-cell Repli-seq experiments in hTERT-RPE1, female mouse embryonic stem cells (CBMS1 mESCs) and 7-day differentiated mESCs. We only deposited single-cell data sets which passed our filtering steps. RNA-Seq: CBMS1 mESCs sample with 3 individual replicates