Fig. 2: Embryonic replication foci patterns, SCNT scRepli-seq and fork/IOD analysis.

a, The EdU-labelling scheme. b, Representative nuclear EdU staining (replication foci) patterns (Methods). c, Exemplary replication foci images. The colour code represents the EdU staining patterns in b. Green, EdU; magenta, histone H3. Scale bar, 10 μm. d, Replication foci pattern dynamics. The colour code is as in b. The numbers (n) of nuclei and embryos are shown. e, scRepli-seq analysis of SCNT embryos, collected at 1 h intervals covering the S phase. f, Binarized whole-S scRepli-seq profiles of SCNT embryos (80 kb bins, 2-somy mode⁵⁹). Each row represents a single cell, ordered by their sampling order or percentage replication score. Averaged scRepli-seq RT (avg scRT) was calculated by averaging S-phase cells. Heterogeneously late-RT regions (hetero late) were regions with an average scRepli-seq RT of <0.50. RT class definitions have been described previously²⁰; CE, constitutively early; CL, constitutively late; D, developmentally regulated. PC1 is the A/B compartment profile based on cumulus cell Hi-C⁶⁰. Embryos were sampled hourly (1 cell, 3–10 h after strontium activation; 2 cell, 1–8 h after cell division). SCNT S-phase lengths were predetermined by EdU. Asterisks indicate data reflecting gradual and uniform replication (Fig. 1d). g, DNA fibre spreading assay. Embryos (early S) or mESCs (control, asynchronous) were labelled with IdU/CldU in vivo followed by fibre extension and immunostaining. h, Representative images of three fork categories. IdU and CldU (green), CldU (magenta) and ssDNA (blue) signals on DNA fibres. Scale bar, 4.55 μm (20 kb). i, DNA fibre classification based on fork categories. j, The IOD between the immobile single-dot forks and IOD on mobile 8-cell fibres (Extended Data Fig. 5b). k, The mobile fork speed. l, G1-to-S-phase lengths revealed by PCNA live-cell imaging. The numbers (n) of cells from a total of 26 embryos are shown. Error bars represent the mean ± s.d. (j–l).