Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a 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 profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication 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, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.

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Data availability

All RT profiles (BrdU-IP, 100 cells and single-cell Repli-seq) and RNA-seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) database under accession code GSE108556.

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We thank S. Kuraku and members of his laboratory for assistance with NGS, F. Matsuzaki for the use of FACS and A. Tanigawa and Y. Kondo for technical assistance. We also thank D. M. Gilbert for exchanging unpublished observations; H. Niwa and K. Araki for CBMS1 mESCs; and B. D. Pope, M. Sugimoto and H. Masai for helpful discussions. This work was supported by a RIKEN CDB/BDR intramural grant to I.H., the Special Postdoctoral Researcher (SPDR) Program of RIKEN to S.T. and a Grant-in-Aid for Scientific Research on Innovative Areas (16H01405) to S-i.T., 18H05530 to I.H., 18K14681 to S.T., 15K06942, 15H01462 and 17H06426 to K.N., from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

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Author notes

  1. These authors contributed equally: Saori Takahashi, Hisashi Miura, Takahiro Shibata.


  1. Laboratory for Developmental Epigenetics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan

    • Saori Takahashi
    • , Hisashi Miura
    •  & Ichiro Hiratani
  2. RIKEN Center for Developmental Biology (CDB), Kobe, Japan

    • Saori Takahashi
    • , Hisashi Miura
    •  & Ichiro Hiratani
  3. Department of Biochemistry and Proteomics, Graduate School of Medicine, Mie University, Tsu, Japan

    • Takahiro Shibata
    • , Masato Ogata
    •  & Shin-ichiro Takebayashi
  4. Laboratory of Molecular & Cellular Biology, Graduate School of Bioresources, Mie University, Tsu, Japan

    • Takahiro Shibata
    •  & Katsuzumi Okumura
  5. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Japan

    • Koji Nagao
    •  & Chikashi Obuse


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S.T., H.M., T.S., S-i.T. and I.H. conceived the project. S.T., T.S. and S-i.T. developed and conducted scRepli-seq and BrdU-IP experiments. S.T. and I.H. performed mESC culture, differentiation and sample collection. T.S. and S-i.T. performed hTERT-RPE1 cell culture and sample collection. K.N. and C.O. constructed diploid reference genome and helped with the haplotype-resolved analysis pipeline setup. H.M. and S-i.T. performed bioinformatics analyses. K.O. and M.O. supported for the design and execution of the project. S.T., H.M., S-i.T. and I.H. wrote the manuscript.

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The authors declare no competing interests.

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Correspondence to Shin-ichiro Takebayashi or Ichiro Hiratani.

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