Extended Data Fig. 3: Details of generation and analysis of single-nucleus RNAseq dataset.

(a) Example plots from FACS sorting of GFP+ nuclei. (b) Plots showing the distribution of cells from either the male or female samples, showing all clusters were represented by both samples. (c) Quality control plots of number of genes (features), UMIs, and percent mitochondrial reads for each sample. (d) Dotplot of classic DA neuron markers as well as Mbp and Gad2, which were used to determine non-classical DA clusters (12, 13, 14 & 15). Clusters 12, 14 & 15 significantly under-express DA neuron markers. Mbp is significantly expressed in cluster 13. Gad2 expression is limited to cluster 8, suggesting this cluster represents a previously described population of gabaergic dopamine neurons. (e) Overlaid expression patterns of Sox6 (green) and Calb1 (red) recapitulates a previously observed dichotomy among midbrain dopamine neurons. (f) Dendrogram of hierarchical clustering estimation. Height of branch points approximates the relatedness of clusters. Notably, clusters 1–4 appear to be Sox6+, 5–7 are Otx2+, and 8–11 are negative for both markers. (g) Heatmap of top 4 differentially expressed genes for each cluster, excluding non-classical DA clusters. (h) Dotplot of expression of key marker genes of dopamine neuron subpopulations.