scRNA-seq analysis identifies two distinct ILC2 subsets in neonatal mouse lungs. (A) Gating strategy for purification of Lin^lo cells from 12-d-old neonatal RORα-YFP mouse lungs for scRNA-seq analysis. (B)t-SNE plot shows distinct clusters within the 21,256 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 clusters from B were combined and subjected to further unsupervised clustering, which divided the 600 ILC2s into subset 1 (293 cells) and subset 2 (307 cells). Heatmap shows 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 our selection of ILC(2)-associated genes by the subsets 1 and 2. The differentially expressed genes are shown by red (higher expression on subset 1) and purple (higher expression on subset 2) boxes. The rest of the genes are similarly expressed by the two subsets.