Fig. 1

Clustering and differential expression analysis reveal substantial heterogeneity of primary culture microglia. a Flowchart of the experimental procedure used to generate data. Brain cortices was extracted from P1 mice. Single cells were dissociated from brain tissue and a glial co-culture was prepared. At the point of confluency, microglia were shaken off, which were used for single-cell barcoding using 10X Genomics 3′ Chromium v3. Libraries were prepared and sequenced, then the data were computationally analyzed with the Seurat package. b UMAP embedding and clustering of 4855 Myeloid cells reveals 7 distinct clusters of primary culture microglia. Data were downsampled to 2000 cells for visualization. c Heatmap showing the expression in each cell of the top differentially expressed genes that mark each cluster. Heatmap contains only the 2000 downsampled cells. d Feature plot of canonical microglia and myeloid markers. Cells are colored based on normalized expression value for each gene. Robust expression of shared myeloid markers is seen. However, expression of microglia-specific markers like Tmem119 is sparse. Violin plots show that the cultured cells robustly express markers of microglial heterogeneity in the developing brain; however, these markers are broadly expressed across many clusters as opposed to in a cluster-specific manner as seen in freshly isolated developing microglia