Extended Data Fig. 4: scRNA-seq characterizes the microglial cross talk with astrocytes and endothelial cells in the aged brain.

(a) Scheme of brain scRNA-seq from young (3 MO) and aged (24 MO) mice. N = 5 mice for each group. (b-c) tSNE plots of 4,326 3 MO and 2,762 24 MO brain cells. Cells are colored according to age (b) and cell type (c) separately. (d) Dot plot showing the expression levels of well-known representative cell-type-enriched marker genes across all 15 cell types (16 populations) (see details in Methods). (e) Dot plot showing the expression levels of two astrocyte subtype marker genes. Gja1, Mfge8, Apoe, Slc1a2 and Slco1c1 were relatively enriched in Astrocyte 1, while S100β, Sox9, Gfap and Aldh1l1 were highly expressed in Astrocyte 2. (f) Strip chart displaying 24 MO vs. 3 MO fold-changes in gene expression levels in each cell population. Colored dots indicate significantly up- or down-regulated (P < 0.05, log₂FC ≥ 0.25) genes in aged cells compared to young cells. Non-parametric Wilcoxon rank sum test. (g) Heat maps of 24-month-old vs. 3-month-old DEGs (significantly up- or down-regulated (P < 0.05, log₂FC ≥ 0.25) in no less than 2 populations) in Astrocyte 1 cells, Astrocyte 2 cells, EC and microglia. (h) Top significantly enriched biological processes of DEGs (significantly up- or down-regulated (P < 0.05, log₂FC ≥ 0.25) annotated by GO (Q < 0.05) in Astrocyte 1, Astrocyte 2, EC and microglia. The accumulative hypergeometric test is used for statistical analysis with false discovery rate (FDR) correction. MO: month-old; GO: Gene Ontology; BP: biological process; EC: endothelial cell; VSMC: vascular smooth muscle cell; OL: oligodendrocyte; OPC: oligodendrocyte precursor cell; ABC: arachnoid barrier cell; NSC: neural stem cell; CPC: choroid plexus epithelial cell; NK: natural killer cell.