Fig. 6

Female cells show delayed onset of differentiation relative to male cells. A Semi-supervised genome-wide trajectory analysis of non-allelic F121 and F123 sci-RNA-seq data with Monocle2 DDRTree using 14,037 differentially expressed genes across time points with q-value ≤ 0.01 and showing expression in at least 10 cells. Arrows indicate the direction of development in pseudotime. Branch 1 is the root ESC branch; branch 2 represents the default embryoid body (EB) pathway; and branch 3 captures NPCs, which derive from d11 cells. Inset table gives cell counts per time point per trajectory branch as well as the time point color used for data points in the plot. B Expression (log10 UMIs) of select genes with respect to pseudotime based on the trajectory in A. Klf4 is a pluripotency factor; Otx2 is transiently expressed during EB differentiation; Sema5a is upregulated during EB differentiation; Map2 is a NPC-specific marker; Xist is upregulated in female cells to initiate XCI. Data points are cells colored by time point (shown above) on the left, and by sex (female pink, male, blue) on the right. C As in A, but based on 13,532 autosomal genes and faceted by cell sex (F121 on the left, F123 on the right). Bar graphs show the proportion of F121 to F123 cells per branch of the trajectory per time point. Compared to male cells, female d3, d7, and d11 cells are enriched along the ESC branch 1, while d3 cells are under-enriched along the EB and NPC branches (branches 2 and 3) indicating a delayed onset of differentiation. Percentages of less than 10% omitted. D Heatmap of genes in aligned kinetic curves where columns correspond to the log-transformed UMIs at each point in the aligned trajectory. These values were further transformed into per-gene Z scores. Each row shows a pluripotency factor gene. E As in C, but for 361 X-linked genes. F UMAP of the non-allelic F121 and F123 sci-RNA-seq count matrix colored by time point (left) and sex (right). G As in F, but colored by the ratio of X-chromosomal to autosomal gene expression (X:A ratio) based on genes that were expressed in at least 10 cells. H As in A, but excluding NPCs and faceted by sex (F121 on the left, F123 on the right). On the right are plots of cumulative distributions of cells’ pseudotimes (rescaled between 0 and 1) along the trajectory arc for female F121 and male F123 cells (see Additional file Fig. S6I, M, Q, K, O, S). I As in H, but based on non-allelic chromatin accessibility (see Additional file 1: Fig. S6G. H, J, N, R, L, P, T)