a, Principal component analysis for each lineage. Only cells that came from embryos that contained all three major lineages were used. In total, 3,145 single cells were included: EPI, n = 282; PE, n = 138; TE, n = 2,725. b, The developmental trajectory of each lineage based on Monocle2. Only cells that came from embryos that contained all three major lineages were used. In total, 3,145 single cells were used. EPI, n = 282; PE, n = 138; TE, n = 2,725. c–e, t-SNE analysis of EPI cells at all four stages revealed three clusters. c, Clusters are shown for each of the days. d, We found that the main reason one minor cluster (in grey) of these three clusters was separated might be owing to the differences in the number of expressed genes, although all individual cells from these clusters passed our quality control in the first procedure of data processing. We could not exclude that this was caused by low transcriptional activity of this cluster of cells or technical limitations in our system. e, We therefore removed this cluster of cells and focused on the differentially expressed genes between those two main clusters (C1 and C2). c–e, In total, 282 single cells were included. f, The results showed that the differentially expressed genes in the population were basically consistent with that in the internal EPI stages, reflecting that the differences in gene-expression characteristics between the two clusters are mainly due to the diversity in developmental stages (days 6 and 8 compared with days 10 and 12), indicating that EPI subgroups mainly reflected the developmental stage-specific differences. The gene list related to f is provided in Supplementary Table 5. g, Principal component analysis for TE at different developmental stages. The sublineages of TE emerge around day 10. Day 6 TE, n = 667 cells; day 10 TE, n = 1,443 cells; days 12 and 14 TE, n = 792 cells (day 12) and 533 cells (day 14).