Extended Data Fig. 4: In-depth scRepli-seq data analysis of SCNT embryos.

(a) Binarized and log2median scRepli-seq profiles of SCNT 1- and 2-cell embryos (80-kb bins), along with BrdU-IP population RT profiles of mESCs. 2-somy mode was applied for binarization. Chr3 is shown. Asterisks, data reflecting gradual and uniform replication (see Fig. 1d legend for details). (b) RT class distribution of all genomic bins and heterogeneously late RT regions (defined as regions with an average scRT of <0.50 from the analysis of S-phase cells) in SCNT 2-cell embryos. The majority of the heterogeneously late RT regions were classified as constitutively late-replicating (CL). RT class definitions are from Dileep et al.²⁰ (CE, constitutively early; CL, constitutively late; D, developmentally regulated). (c) A/B compartment (Hi-C PC1) category distribution of all genomic bins and heterogeneously late RT regions on the SCNT 2-cell genome. A1 (strongest A), A2, B2, and B1 (strongest B) each contain 25 % of all PC1 values (200-kb bins) based on a cumulus cell Hi-C data⁶⁰. More than 80% of the heterogeneously late RT regions were classified as B (heterochromatin) compartment, B1 and B2. (d) Averaged S-phase scRepli-seq profiles and heterogeneously late RT regions on the 1-cell paternal genome and in SCNT 2-cell embryos, along with BrdU-IP population RT profiles of mESCs and RT class distribution (see also Fig. 2f and Extended Data Fig. 2h). There is a substantial overlap between the heterogeneously late RT regions on the 1-cell paternal genome and in SCNT 2-cell embryos just by visual inspection. Chr12 and chr4 are shown. (e) A Venn diagram showing the degree of overlap between the heterogeneously late RT regions on the 1-cell paternal genome and the SCNT 2-cell genome (80-kb bins). (f) MAD score distribution¹⁶ of SCNT 1- and 2-cell embryos analysed by scRepli-seq. SCNT 1-cell embryos showed constant MAD scores throughout S-phase as in control 1- and 2-cell embryos (Fig. 1e and Extended Data Fig. 2f,g). In contrast, while the SCNT 2-cell embryos did not show inverted V-shape patterns observed in mESCs and control 4-cell embryos (Fig. 1e), they clearly showed fluctuation during S-phase, indicative of the co-existence of ~2 (nearly replicated) and ~1 copy (under-replicated) genomic bins.