Identification of surface molecules for sorting of iNKT sublineages using scRNA-seq. (A) Uniform manifold approximation and projection (UMAP) plot of scRNA-seq data showing distinct clusters of iNKT cell subsets: iNKT1 (blue), iNKT2 (red), and iNKT17 (green) cells. (B) Dot-plot showing the proportion of cells (dot size) and the scaled (z score) gene expression of genes encoding for the iNKT sublineage-defining transcription factors T-Bet (Tbx21), PLZF (Zbtb16), and RORγT (Rorc). (C) Single‐cell heatmap representing the 10 most highly differentially expressed genes in thymic iNKT cell subsets. Expression for each gene is scaled (z scored) across single cells. (D) Normalized counts of Icos, Pdcd1, and Cd4 RNA expression. (E) FACS-sorting strategy for isolation of iNKT sublineages based on surface molecules starting from CD19^–, CD8a^–, CD62L^–, TCRγδ^–, GR-1^–, Ter119^–, and CD24^– cells. Cell purity of FACS-sorted iNKT sublineages assessed by intranuclear staining for the transcription factors PLZF and RORγT. (F) Heatmap representing the 10 most highly differentially expressed genes identified in scRNA-seq analysis in bulk RNA-seq analysis performed on sorted populations. Expression for each gene is scaled (z scored) across single rows.