Supplementary Figure 3: Validation of microglia-specific TRAP mice.

(a) Schematic showing TRAP-sequencing from microglia-specific and astrocyte-specific TRAP mice. (b) Volcano plot for differential expression analysis (DESeq2, n=2/genotype/region) of each gene. y-axis: -log10 (adjusted p-value), and x-axis: log2 (fold change) between astrocyte- and microglia-TRAP. 885 genes (green) are significantly enriched in microglia TRAP; 970 genes (blue) are significantly enriched in astrocyte TRAP. (c) Heatmap with hierarchical clustering distances shows the variation in the expression levels (z-scored log2 RPKM) of 885 microglia genes and 970 astrocyte genes. (d) Selected Gene Ontology (GO) annotations enriched (Enrichr) for 885 microglia-enriched genes (green) and 970 astrocyte-enriched genes (blue). y-axis: -log10 (p-value). Dotted lines: p-value=0.05. (e) Top 50 microglia genes and top 50 astrocyte genes ranked by highest expression, listed in bar graph with RPKM of each gene (y-axis) in microglia- (green) and astrocyte-TRAP (blue). Bar graphs show mean ± SEM (n=4/genotype). (f) cbMg and stMg are heterogeneous independent of the specific microglia-TRAP mouse line used. Principal component analysis (PCA) based on the expression of top 500 differentially expressed genes of TRAP from the striatum and cerebellum of n=6 Cx3cr1^CreErt2/+;Eef1a1^{LSLeGFPL10a/+} (circles) and n=2 Tg(Prox1-Cre)SJ39; Eef1a1^{LSLeGFPL10a/+} (squares) mice and from the striatum and cerebellum of n=2 Tg(Aldh1l1-eGFP/L10a) astrocyte-specific TRAP mice is shown. (g) Enrichment of microglia-specific genes in microglia-specific TRAP vs astrocyte-specific TRAP or TRAP from wild-type controls. Left: Heatmap with hierarchical clustering distances shows the variation in the expression levels of 885 microglia-enriched genes in TRAP samples from the cerebellum and striatum of Cx3cr1^CreErt2/+;Eef1a1^{LSLeGFPL10a/+}, Tg(Prox1-Cre)SJ39; Eef1a1^{LSLeGFPL10a/+}, Tg(Aldh1l1-eGFP/L10a), and wild-type control mice. Right: Box-and-whisker plots show relative expression of these genes in pooled TRAP samples from the two microglia-specific TRAP lines, Cx3cr1^CreErt2/+;Eef1a1^{LSLeGFPL10a/+} (n=12) and Tg(Prox1-Cre)SJ39;Eef1a1^{LSLeGFPL10a/+} (n=4), from astrocyte-specific TRAP line, Tg(Aldh1l1-eGFP/L10a) (n=4), and wild-type control mice to assess/exclude nonspecific mRNA binding (n=3). Cx3cr1^CreErt2/+: min=0.17, 25%=0.97, median=1.42, 75%=1.80, max=2.83; Tg(Prox1-Cre)SJ39: min=-0.58, 25%=0.66, median=1.12, 75%=1.53, max= 2.99 Tg(Aldh1l1): min=-6.12, 25%=-4.22, median=-3.41, 75%=-2.31, max=-0.91; ctrl: min=-8.48, 25%=-5.12, median=-3.87, 75%=-2.20, max=0.025; p<0.0001, F=3888, Kruskal-Wallis statistic (KWS)=2166, One-way ANOVA (Kruskal-Wallis test) with Dunn’s Multiple Comparison post-hoc analysis to compare all pairs of columns. Box-and-whisker plots (Tukey) and heatmaps show normalized expression values (z-scored log2 RPKM). ***p ≤ 0.001.