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. 2022 Dec 15;220(2):e20220605. doi: 10.1084/jem.20220605

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© 2022 Han et al.

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Figure S1. — Quality control of single-cell sequencing of microglial response to IL-33. (A) Gating strategy for isolation by FACS of all CD45⁺ (red) for scRNA-seq in Fig. 1, or microglia only (CD11b⁺, CD45^low, Ly-6C⁻, violet) for bulk RNA-/ATAC-/ChIP-seq (Fig. 2). (B) Gating strategy showing microglia (CD45^lowCD11b⁺⁾, myeloid (CD45^highCD11b⁺), and lymphoid cells (CD45⁺CD11b⁻) after vehicle or IL-33, gated on CD11b and CD45. Representative plots from thalamus, quantifications include thalamus and cortex. Dots = mice. (C) Violin plots of scRNA-seq data showing transcripts/cell, unique genes/cell, and percentage mitochondrial content/cell for each sample. Cut-off boundaries are marked (upper: red line; bottom: black line). (D) Feature plots for the number of genes and percentage mitochondrial RNA for all samples combined from scRNA-seq data. Each dot represents a cell. (E) Feature plot showing correlation between two replicates used for WT + IL-33 scRNA-seq sample. (F) Heatmap for the top three genes in each cluster from scRNA-seq data. (G and H) Mean fluorescence intensity for MARCO protein in cortex from Cx3cr1^creERT2 (G) and P2ry12-^creERT2 (H) mice. Dots = individual mice. Two-way ANOVA with Tukey’s post hoc comparison (genotype and treatment). (I and J) Percentage of microglia expressing MARCO protein in thalamus from Cx3cr1^creERT2 (I) and P2ry12-^creERT2 (J) mice. Dots = individual mice. Two-way ANOVA with Tukey’s post hoc comparison (genotype and treatment). (K and L) Percentage of microglia expressing TLR2 protein in thalamus from Cx3cr1^creERT2 (K) and P2ry12-^creERT2 (L) mice. Dots = individual mice. Two-way ANOVA with Tukey’s post hoc comparison (genotype and treatment). (M and N) Representative images for P2ry12^creERT2 crossed to a R26R-TdTomato (TdT) reporter (Ai14). (M) Top row shows cortex with overlying meninges. Arrowheads indicate meningeal macrophages that are CD68⁺IBA1⁺TdT⁻. Bottom row shows L2/3 cortex. Arrowhead indicates perivascular macrophage that is CD68⁺IBA1⁺TdT⁻. (N) Ventral posteromedial (VPM; top) and ventral posterolateral (VPL; bottom) nuclei of thalamus. Arrowhead indicates perivascular macrophage that is CD68⁺IBA1⁺TdT⁻. Scale bar = 50 μm. (O and P) Representative flow plots of cortex (O) and thalamus (P) showing P2ry12^creERT2 driven Ai14 (TdTomato) expression at P15. Two gating strategies shown: Left panel shows that of Ai14⁺ cells, 99.9% fell within the CD45/CD11b microglial gate. Right panels pre-gate on microglia, myeloid, and lymphoid cells based on CD11b and CD45 followed by Ly-6C and Ai14, to show the percentage of each population with detectable TdT. (Q) qRT-PCR of Il1rl1 expression in cortical and thalamic microglia comparing Cx3cr1^creERT2 and P2ry12^creERT2 mice. Values normalized to housekeeper (Hmbs) and then control + vehicle (PBS) condition. In Cx3cr1^creERT2 the control = Cx3cr1^creERT2+/− + vehicle (PBS). In P2ry12^creERT2 the control = Il1rl1^fl/fl + vehicle (PBS). Each dot represents a mouse. Two-way ANOVA followed by Tukey’s post hoc comparison (genotype and treatment). Data points represent the average of three technical repeats for Cx3cr1^creERT and two technical repeats for P2ry12^creERT. Data represented as mean ± SEM for bar graphs. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.