Extended Data Fig. 1: Evaluation of scRepli-seq resolution and the analysis of scRepli-seq profiles of pre-implantation mouse embryos.

(a) NGS tag density plots of mid-S mESC scRepli-seq data (three individual cells) with 4–6 million (M) (top panel) and ~20 M (bottom panel) total NGS reads per cell. Density was calculated using sliding windows of 200 kb at 40-kb intervals, 80 kb at 16-kb intervals, 40 kb at 8-kb intervals, or 20 kb at 4-kb intervals. While the bimodal distribution is evident when using 200-kb and 80-kb windows, it becomes obscure when using 40-kb windows. For the ~20 M read samples, bimodality is barely visible at 40-kb. (b) scRepli-seq NGS read density plots of representative mid-S mESC single-cell data (Extended Data Fig. 1a, MidS#1) with 4.6 M and 18.4 M reads with the indicated bin size settings. Binarization calling outputs are also shown. Note that the valid read count is not necessarily proportional to the total read count, owing to an increase in duplicate reads, suggesting the presence of redundancy due to the limit of the scRepli-seq library complexity. The dotted line shows the lower read count limit of 20 that we set for the unreplicated bins (see Supplementary Note 1). (c) Pearson correlation matrix of averaged mid-S cell scRepli-seq data (excluding chrX). (d) Average scRepli-seq RT (Ave. scRT) profiles calculated from mid-S cells with 30–70% replication scores derived from 8-cell embryos and ICM. Chr8 is shown (80-kb bins). RT class definitions are from Dileep et al.²⁰ (CE, constitutively early; CL, constitutively late; D, developmentally regulated). RT-switching regions were defined as regions with ∆ave. scRT (ICM–8-cell) values of >1 or <–1. (e) Types and frequency of RT switching (from the 8-cell stage to ICM) and their RT classes (defined in (c)). LtoE and EtoL represent RT changes from late-to-early and early-to-late S, respectively. (f) Pearson correlation matrix of averaged scRepli-seq data of ICM and TE derived from mid-S cells (30–70% replication score, excluding chrX). (g) Haplotype-resolved binarized and log2median scRepli-seq profiles of 1/2/4-cell B6MSM embryos along with BrdU-IP population RT profiles of mESCs. For binarization by AneuFinder⁵⁹, 2-somy mode was applied to 1-, 2- and mid/late 4-cell blastomeres, and 1-somy mode to early 4-cell blastomeres. 1- and 2-cell scRepli-seq profiles are ordered based on their sampling order, as we were unable to calculate their percentage replication score values. The resolution of binarized RT profiles and log2median RT profiles of non-haplotype-resolved B6MSM data are 80 kb (non-overlapping windows) and 200 kb (sliding windows at 40-kb intervals), respectively. For B6 or MSM (haplotype-resolved) data, it was 400 kb (non-overlapping windows) and 1 Mb (sliding windows at 40-kb intervals), respectively. Heterogeneously late RT regions shown beneath the 1-cell scRepli-seq profiles were defined as regions with an average scRT of <0.75 on the MSM (paternal) chromosome. Asterisks, data reflecting gradual and uniform replication (see Fig. 1d legend for details).