Fig. 1. Single-cell RNA sequencing reveals that Clec4a2 expression defines vascular macrophages with homeostatic features in the murine atherosclerotic aorta.

a Gating strategy for CD45⁺CD11b⁺ cells from whole aortas of 20–26-week-old ApoE^−/− female mice fed a high-fat diet (HFD) for 12–16 weeks. b ivis clustering of 16,100 vascular myeloid cells analysed by single-cell RNA sequencing (scRNA-seq) using the 10X genomics platform. Cells were pooled from 9 mice in each experiment. Three independent experiments were performed and normalised before concatenation. c Heatmap of differentially expressed genes (P < 0.05) in 13 clusters. Cluster 12 expressing stromal genes was excluded from further data analysis. Differentially expressed genes were identified using the two-sided Wilcoxon Rank Sum test with Bonferroni’s correction for multiple-hypothesis testing. d Violin plots of selected genes in 12 myeloid clusters. e The Gene Ontology (GO) enrichment analysis of clusters based on differentially expressed genes in resident-like macrophage subsets (C0, C1, and C5) and C6. Enriched pathways are presented as -Log₁₀ (P values) using topGO analysis. P values were obtained using the one-sided Fisher’s exact test without multiple-testing correction. P values < 0.05 were considered significant.