Extended Data Fig. 3: RNA sequencing of Dicer KO microglia from PS19 mice.

(a) Volcano plot of RNA-seq data from Dicer KO microglia from 3-month-old male and female PS19 mice. Pink, female-enriched; turquoise, male-enriched; grey, not significantly different. Vertical dashed lines indicate log2FC ± 1. Horizontal dashed line indicates -log10(0.05). n = 4 male samples, 2 female samples, 2 mice/sample. Wald test was used. (b) Schematic of the single-cell isolation method. Brains without the cerebellum were harvested from 9-month-old Dicer KO PS19 female and male mice and homogenized. After myelin depletion, cells were sorted using flow cytometry and gated by CD45⁺;CD11b⁺ expression. (c) Representative FACS plots showing gating strategy and the cells sequenced. Similar gating strategy was used for all samples sequenced (n = 2 biologically independent animals/sex). (d) Number of cells, proportion and statistics for FACS plots from (c). (e-g) Quality control criteria for the single-cell sequencing data. Fitted curves for histograms of mapped reads (e), numbers of detected genes (f) and ERCC correlation coefficient (g) are labeled in red. Dashed lines are statistical cutoffs. Cells that passed all three criteria were retained for analysis. (h) Scatter plot highlighting cells that passed QC (red) among all cells sequenced. Each dot is a cell. (i) Summary of the numbers of cells sequenced and cells that passed QC (red). (j) t-SNE plot of microglia clusters from 9-month-old Dicer KO PS19 female and male mice. n = 2 biologically independent animals/sex. (k) Heatmap of top genes defining each microglial cluster. (l) Ridge plots of microglial marker expression levels by each microglial cluster.