Extended Data Fig. 3: Replication foci and H3K9me2 dynamics during early cleavage stages.

(a) (top) Experimental scheme. (left) A cell-cycle fluorescence-activated cell sorting (FACS) profile of mESCs. Cells were stained with Hoechst 33342, and the six gates (S1 through S6) used to collect cells throughout S-phase are shown. (right) Replication foci pattern dynamics throughout S-phase (this figure is identical to Fig. 2d). Replication foci patterns were categorized into the following classes: G1, no signal; I, uniform (weak signal); II, uniform (strong signal); III, nuclear periphery + internal foci; IV, internal foci. (b, c) scRepli-seq profiling after replication foci image analysis. (left) Replication foci pattern classification based on the definition provided in (a) of 4-cell (b) and 8-cell (c) embryos. M, mitosis. (right) Binarized scRepli-seq profiles of the 20 (b) or 10 (c) EdU-stained nuclei shown on the left. Our data provide direct, single-cell-level evidence that the spatial patterns of replication foci are a good indicator of S-phase time. Chr2 is shown. For experimental details, see Methods under ‘Single-cell DNA replication profiling (scRepli-seq) using mouse embryos’. (d) Top panel shows the representative images of H3K9me2 (green) staining. Histone H3 was stained for reference (magenta). Bottom panel shows the heatmap images of the H3K9me2 signal. (e) H3K9me2 levels were increased after 4-cell stage. One-way ANOVA using Dunn’s multiple comparisons test was performed. Error bars represent the mean and SD. (f) The spatial pattern of H3K9me2 shifted at the transition from 2-cell to 4-cell stage. Number of nuclei from two independent experiments are shown in (e) and (f).