Supplementary Figure 4: Microglia-specific TRAP analysis reveals region-specific expression patterns and prevents ex vivo isolation-induced activation of microglia.

(a) Schematic showing FACS-based microglia isolation performed in parallel with microglia-specific TRAP-seq. (b) Left: Representative flow cytometry data illustrates isolation of Percoll-enriched microglia (Methods) from the striatum / cerebellum of Cx3cr1^CreErt2/+; Eef1a1^{LSLeGFPL10a/+} mice. x-axis: GFP signal, y-axis: Aqua Live/Dead signal to select for live cells. Gate for GFP-positive cells. Percentage of the populations gated compared to total scatter is shown. Right: Quantification of percentages of GFP+ events in the striatum (mean=50.23, SEM=4.491) and cerebellum (mean=40.97, SEM=7.441) p=0.0928, F=2.746, t₂=3.050. Bar graphs with individual data points show mean ± SEM. Two-tailed paired t-test (n=3 independent experiments). (c) mRNAs obtained by TRAP and by microglia isolation display a high correlation by linear (Pearson) correlation. Representative scatter plots of genic expression with correlation values between individual (n=1 replicate) TRAP and FACS samples from stMg and cbMg. Scatter plots of genic expression were reproduced with similar results for all replicates. (d) MA plot for differential expression analysis (DESeq2, n=2/region/method) of each gene shows log2 (mean expression) across all samples (x-axis) vs log2 (fold change) by FACS over TRAP (y-axis). 683 genes (red) are significantly induced by FACS. Genes in blue are down-regulated by FACS and genes in green are equally enriched. (e) Heatmap shows the variation in the expression levels (z-scored log2 RPKM) of 40 pro-inflammatory and immediate early genes in FACS and TRAP samples. (f) Genome browser views (IGV) of selected genes in TRAP and FACS samples.