(a) Experimental design for microglia depletion and repopulation. Mice were placed on PLX diet (1200 mg/kg) for 14 days to deplete microglia (D0). The early stage microglial repopulation (D2) group was switched to control diet (CD) for 2 days. Microglia from each mouse were collected on the same day. A total of 15 female C57BL/6J mice (5 Mo) were used: Ctrl (n = 3); D0 (n = 6, two brains pooled together for FACS); D2 (n = 4, two brains pooled together for FACS); (b) Workflow of adult microglia isolation procedures for scRNA-seq capture. Detailed description in methods. (c) Heatmap showing the top 180 variable genes detected from 28,649 cells after initial data filtering. (d) UMAP plot showing nine distinctive clusters identified from the scRNA-seq data. (e–h) Violin plot showing expression of Itgam, Aif1, Cx3cr1, and Csf1r in all clusters. Clusters 6, 7, 8, and 9 were removed from downstream data analysis. (i) Reclustered UMAP after removal of clusters 6–9 showing five distinctive clusters. (j) UMAP split by experimental conditions. (k) Ratio of cells from three treatment groups distributed in each cluster. Ratio of cells was calculated by normalizing to the total number of cells captured in each sample (n = 3 for Ctrl and D0, two for D2). Data shown as mean ± SEM. p-values were calculated using the negative binomial generalized linear model from EdgeR. *p≤0.05; ***p≤0.001.