Extended Data Fig. 7: Genome-wide analysis of replication timing (RT) in the transitional S-phase during which 2CLC emerge.

a. S-phase length in mother cells of 2CLCs and ESCs and in ESCs and 2CLCs during the transitional S-phase. Boxplots: median (middle line) IQR (boxes) and extent of data without outliers (whiskers,>1.5x IQR). Notches extend to + /−1.58xIQR/sqrt(n), indicating confidence intervals. Dots are individual measurements arranged in 0.2 h bins. b. Scatter plots of read density at 100 Kbp bins across the genome between Repli-seq replicates of the transitional S-phase of cells transitioning from ESC to 2CLC. Pearson R² is indicated. c. Pearson R correlation heatmap based on read density at 100 Kbp bins across the genome in each S-phase of cells transitioning from ESC to 2CLC. d. Pie charts of numbers of genes that replicate in early, mid and late S-phase in ESCs, for gene-sets whose replication shifted to earlier and later timing during the transitional S-phase in emerging 2CLCs. e. Enrichment of repeat elements across genomic regions changing to an earlier and later replication timing during the transitional S-phase at which 2CLCs emerge. f. H3.3 enrichment at MERVL-int and MT2_Mm repeats in 2-cell embryos. Reads were normalized by sequencing depth and length, data from two biological replicates shown separately as 25th and 75th percentiles (box), median (line) and smallest and largest values within 1.5×IQR of the hinge (whiskers). Statistical analyses against the input were with two-sided Wilcoxon-signed-rank test. g. Developmental progression of fertilized embryos upon HU treatment. Zygotes collected at 17-18 h post-hCG were treated with HU until 48 h posthCG. Embryos reaching the blastocyst stage (%) are indicated; n: number of embryos analyzed. Scale bar, 100 μm. h. RNAseq quality control (QC) metrics for nuclear transferred embryos (control and 10μM HU-treated) and single cumulus cells. QC thresholds (red dotted lines) are indicated; samples failing QC (triangles) were discarded. Boxplots show median and IQR; whiskers depict the smallest and largest values within 1.5×IQR. i. Heatmap with expression of ZGA genes upon nuclear transfer compared to in vivo derived embryos. j. Cell death analysis by dual Annexin-V and propidium iodide (PI) staining following HU treatment. Cells positive for either or both Annexin-V and PI were considered dead. Statistical analyses: two-sided Student’s t-test.