Extended Data Fig. 4. scRNA-seq analysis of hepatic ILC1 from WT and Hobit^KO mice.

a Schematic representation of the Hobit locus of Hobit^KO mice. b Absolute numbers of liver NK cells of WT and Hobit^KO mice. c Representative FACS analysis displaying Lin⁻NK1.1⁺ hepatic ILC1 and NK cells in Hobit^Tom/WT and Hobit^Tom/KO mice. Bar graphs indicate absolute numbers of liver ILC1. Data in (b) and (c) are pooled from 2 independent experiments with n=3 (b) and n=2 and 3 (c) mice per group. d Representative FACS analysis of liver ILC1 of Hobit^KO x RORc-eYFP fate mapper (FM) mice. Bar graph shows frequency of RORc fate map-positive cells within CD127⁺ and CD127⁻ liver ILC1. Data are representative of 2 independent experiments with n=2-3 mice per group. e-g scRNA-seq of hash-tagged liver ILC1 sorted from Hobit^Tom/WT and Hobit^Tom/KO mice. e General QC, low quality cells (feature counts > 1000 and percentage of genes mapped to mitochondrial genome < 8) and cell doublets filtering and demultiplexing of Hashtag (HT) antibodies allows to separate Hobit^KO/Tom (KO) and Hobit^Tom/WT (WT). Single cell transcriptome visualization using a UMAP color-coded by clusters and the sample WT or KO as indicated by the binary detection of the HT Abs. f Volcano plot displaying significantly differentially expressed genes between WT and Hobit^KO ILC1, using Wilcoxon test. Thresholds are displayed at Log2 FC equal 0.5 and Adjusted P value of 0.05. Genes on the right are overexpressed in KO, genes on the left are overexpressed in WT. g Heat map of marker gene expression differentially expressed across liver ILC1s. Cluster number refers to panel (e). h, i Pseudo-temporal ordering of ILC1 single cell transcriptomes with Slingshot. Expression of candidate genes across pseudotime within hash-tagged liver ILC1s sorted from Hobit^Tom/WT and Hobit^Tom/KO mice. Color barcode in (h) indicates cluster identity (as identified in e). Heatmap of expression of selected genes in ILC1 populations along pseudotime (i). Bar graphs indicate individual mice (symbols) and mean (bar), error bars display means ± SD. Statistical significance was calculated by unpaired two-tailed t-test; **p < 0.01, ns – not significant.