Fig. 4

Little overlap between in vitro and freshly isolated microglia transcriptomes. a Schematic diagram of microglia isolation and analysis methods. Seurat canonical correlation analysis (CCA) integration was used to perform clustering and UMAP embedding on the combined cell population. b Seurat clustered the 13,817 combined cells (8794 freshly isolated cells, 4855 astro-plated in vitro cells, 168 micro-isolated in vitro cells) into 10 populations that show heterogeneity in the UMAP. Cells were downsampled to 2000 for visualization. Astro-plated and micro-isolated cells analyzed in combined UMAP are the same cells analyzed in Fig. 1 and 2. c UMAP of the combined cells colored by cell source. Even after CCA integration, distinct separation between microglia from in vitro and freshly isolated sources is apparent. d Colored UMAP embedding labeling the cells by previously determined clusters from Fig. 2. e Bar graph quantifying the percentage of each Seurat cluster that is made up by cells from the 3 different sources. The majority of clusters are close to 100% represented by cells from a single source, though the 4 highlighted clusters—3,4,7, and 8— originate from mixed sources. f Subclustering of the mixed-source cluster 3, highlighting the two largest representative clusters: astro-plated microglia-like cluster 1 and freshly isolated activated cluster. Pie chart shows the proportion of cells in this integrated cluster that are from the different transferred clusters: the two largest components of the cluster are astro-plated micro-like 1 and freshly isolated activated cells, with a minority of cells from other groups including astro-plated macro-like. The 10 top up- and downregulated markers are plotted in a heatmap. g The same plots as subpanel f, but for integrated cluster 4, highlighting the freshly isolated homeostatic cluster and the in vitro microglia-like 3 cluster. Minority components of the pie chart include astro-plated micro-like 1. h The same plots as f, but for integrated cluster 7, highlighting astro-plated mono-like cluster 1 and micro-isolated in vitro cells. Minority cell types of the cluster include astro-plated micro-like 3 and astro-plated micro-like 2 cells. i The same plots as subpanel f, but for cluster 8, which is comprised mostly of the astro-plated monocyte-like 2 cluster and the freshly isolated interferon cluster. Minority cell types of the cluster include astro-plated macro-like and astro-plated micro-like 1 cells