Supplementary Figure 1: Gene-expression analysis of cardiac macrophage clusters defined by single-cell RNA sequencing reveals five distinct populations.

a) Cardiac mononuclear phagocytes were sorted from adult c57bl/6j mice (~20 weeks old). Total macrophages (MFs) (R2; CD45⁺CD64^lo–hiCD11b⁺) and total dendritic cells (DCs) (R3; CD45⁺CD64^–CD11c⁺MHC-II^int–hi). The two sorted populations were pooled in a 1:1 ratio before processing for scRNA-seq on the 10x Genomics Chromium platform. N=8 (4 male; 4 female); one experiment. b) The top ~65 differentially expressed genes in each cluster were input into the ImmGEN online portal for "My Geneset" http://rstats.immgen.org/MyGeneSet_New/index.html to generate a similarity score. We included all MF, monocyte and dendritic cell populations in the V1 array database for the assessment to generate a means-normalized expression scatter plot. Colored boxes highlight similarity in dendritic cell (light green), macrophage (dark green) and monocyte (red) populations to the sorted populations contributed to ImmGen. c) Violin plots of cluster defining genes in the 11 clusters defined in Fig. 1c. *p<0.01 (adjusted P value). Statistical analysis of scRNA-seq is defined in the material and methods, and was performed using the MAST method.