Fig. 3

The majority of freshly isolated microglia are homeostatic with minor activated and interferon subpopulations. a Schematic diagram of the cell isolation and scRNAseq procedure used to generate this data. b UMAP and clustering of 8794 microglial cells. Clusters 0,1,2 and 3 appear to form a homogenous blob, while the much smaller clusters 4 and 5 are distinctly separate. 2000 downsampled cells are shown for visualization purposes. c Heatmap of the top differentially expressed genes in each cluster. Clusters 0-3 show higher expression of resting microglia markers like Tmem119, cluster 4 shows expression of canonical activation markers like Ccl4, and cluster 5 shows expression of interferon-related genes like Ifit3. d Informed by the differential expression analysis, cells are labeled as either homeostatic, activated, or interferon microglia and replotted on the UMAP embedding. e Violin plots showing 3 canonical markers of homeostatic, activated, and interferon-response microglia validate our classification of cells into labeled clusters. Act. = Activated, Hom. = Homeostatic, Ifn. = Interferon. f To validate the activated microglia cluster represents the same disease-associated microglia signature (DAM) found in mouse models of neurodegeneration, a gene set analysis was run using previously published gene sets known to be changed during activation. Percentage of UMIs belonging to genes in each gene set were calculated for each cell. Ridged box plots show the distribution of percentages between each group. A two-tailed t-test was used to compare gene set percentages between the activated cluster and the homeostatic cluster, and between the interferon cluster and the homeostatic cluster. Bonferroni correction was applied to resulting p-values to control for multiple testing. P-values and statistics from this test are presented in Table 2