ScRNA-seq analysis of adult RORα-YFP mouse lungs identifies two distinct ILC2 subsets. (A) Gating strategy for the purification of Lin^lo cells from adult RORα-YFP mouse lungs for scRNA-seq analysis. (B)t-SNE plot shows distinct clusters within the 4,664 sequenced cells. (C) Heatmap of the differentially expressed genes by the clusters from B. Top 10 differentially expressed transcripts of the lymphoid lineages are shown. (D) The ILC2 cluster from B was subjected to further unsupervised clustering that divided the 371 ILC2s into subset 1 (233 cells) and subset 2 (138 cells). Heatmap shows the top 20 genes that are differentially expressed by the subsets 1 and 2. The arrows point to the genes that are mentioned in the text. (E) Heatmap shows the expression of our selection of ILC(2)-associated genes by subsets 1 and 2. This gene list includes the transcription factors, cell surface receptors, and effector cytokines important for ILC(2) lineage development and function based on the literature. Hence, it includes genes that are equally expressed as well as genes that are differentially expressed by the two subsets. The differentially expressed genes are shown by black (higher expression on subset 1) and green (higher expression on subset 2) boxes. The rest of the genes are similarly expressed by the two subsets.