Figure 2. Single-nucleus RNA sequencing distinguishes distinct cell types and molecular signatures of OB projection neurons.

(A) UMAP representation of gene expression profiles of 31,703 single nuclei combined from all replicates (R1, R2, R3) of mice injected into both AON and PCx, grouped into 22 clusters color-coded by cell type membership (GC: granule cell, PG: periglomerular cell, OPC: oligodendrocyte precursor cell, Micro: microglia, Astro: astrocyte, Oligo: oligodendrocyte, ET: external tufted cell, M: mitral cell, T: tufted cell, Imm-like: Immature-like cell). See Figure 2—figure supplement 1 for detailed quality check of each replicate. (B) Matrix plot showing the z-scored expression levels of the top 50 differentially expressed (DE) genes for each cell population ordered by hierarchical relationships between distinct clusters. Each column represents the average expression level of a gene in a given cluster, color-coded by the UMAP cluster membership (from A). The dendrogram depicts the hierarchical relationships and is computed from the PCA representation of the data using Pearson correlation as distance measure and link by complete linkage. (C) UMAP representations of known marker genes for main cell populations (Syt1: neurons; Gad2: GABAergic neurons; Slc17a7: glutamatergic neurons; Gfap, Tmem119, Pdgfra, Mobp: glial cells; Th, Calb1: periglomerular neurons; Cdhr1, Tbx21, Slc17a6: mitral/tufted cells). Nuclei are color-coded by the raw expression level of each transcript. (D) UMAP representation of subclustering from the initial clusters M1, M2/M3, T1, ET1, and ET2 (cluster names from A), selected for the expression of known excitatory and mitral/tufted cell markers (shown in C), resulting in 7504 putative projection neurons grouped into nine distinct types. (E) Same matrix plot as described in (B) showing the z-scored expression levels of the top 50 DE genes for each projection neuron type ordered by hierarchical relationships and color-coded by the UMAP subcluster membership (from D).

Figure 2—figure supplement 1. Quality check of individual replicates of sn-RNA seq (sn-R1/R2/R3 dataset) shows the reliability of the data and the replicability of each cell type.

(A) Depiction of nuclei for each replicate (in red R1, in green R2, in blue R3) embedded in the UMAP space showing that replicates are very similar to each other and can be combined for downstream analyses. Replicates were merged using Canonical Correlation Analysis (CCA). (B) Bar chart showing the fraction of each cluster when R1, 2 and 3 are combined, color-coded by the replicate membership, indicating that each cell type is consistently represented in each replicate. (C) Scatter plots of pairwise comparisons of log10 gene expression levels averaged over nuclei. In the three panels, replicates R1, R2, and R3 are compared. Pearson correlations are shown in the top left corner of each plot.