Transcriptional analysis by scRNA-seq of primitive, definitive, and FL-derived RBCs. (A) Schematic outline of scRNA-seq experiment. (B) Flow cytometry histograms of erythroid markers at the time of library preparation in primitive (pink), definitive (green), and FL-derived (blue) RBCs. Negative control is shown in gray. (C) Uniform manifold approximation and projection of primitive (n = 9547), definitive (n = 8808), and FL-derived (n = 11 598) RBCs. Subsets of individual samples are shown on the left (primitive iRBCs, pink; definitive iRBCs, green; and FL-derived RBCs, blue). (D) Distinct clusters of primitive, definitive, and FL-derived RBCs as defined by Seurat. (E) Dot plot showing expression of selected marker genes in each cluster. The dot size represents the percentage of cells within a cell cluster in which the gene was detected, and the dot color intensity represents the relative normalized average expression level of that marker. (F) Differential expression of a selection of erythroid genes in primitive, definitive, and FL-derived RBCs. (G) Correlation heat map of FL and definitive and primitive-derived RBCs in erythroid clusters. (H) Histogram showing the percentage of cells in each erythroid clusters in different samples. (I) Heat map of the top 25 differentially expressed genes between primitive and FL-definitive clusters.