Extended Data Fig. 4: Correspondence between scRNA-seq transcriptomic clusters, projection targets, and bulk RNA-seq data.
From: Distinct descending motor cortex pathways and their roles in movement
a, Pairwise correlations (Pearson’s r) in gene expression between all cells in the Slco2a1 cluster and within subsets of Slco2a1 cells identified by projection target. Expression patterns of cells identified from a common target were not more similar than randomly chosen cells. b, As in a for the Hpgd expression cluster. c, Pairwise correlation in gene expression of cells retrogradely labelled from the superior colliculus within the Slco2a1 cluster, within the Hpgd cluster, and between cells from different clusters. Correlations between cells from different clusters were significantly lower than within-cluster correlations (between Slco2a1, superior colliculus and Hpgd, superior colliculus (n = 1,677 pairs) versus within Slco2a1, superior colliculus (n = 903 pairs): P < 1 × 10−10; between Slco2a1, superior colliculus and Hpgd, superior colliculus (n = 1,677 pairs) versus within Hpgd, superior colliculus (n = 741 pairs): P < 1 × 10−10; two-sided Wilcoxon signed rank test). Correlation analysis was not performed for the Npsr1 cluster as the number of cells was relatively small (Fig. 2e). d, Number of differentially expressed genes in bulk RNA-seq data between groups of cells labelled from different projection targets. Notably, only a single gene was identified as differentially expressed between the thalamus/medulla group and the superior colliculus/pons group, indicating that the set of PT neurons projecting to either the thalamus or medulla probably represents a superset of PT neurons. There is unlikely to be a transcriptomically distinct group of PT neurons with projections to the superior colliculus or pons that lacks projections to the medulla and thalamus. Although we cannot rule out a transcriptomically distinct subset that lacks projections to all of the thalamus, medulla, pons and superior colliculus, such neurons were not detected in single-cell reconstructions. e, Total genes detected by bulk RNA-seq and scRNA-seq in PTupper neurons. Both: genes detected in bulk RNA-seq and scRNA-seq; single-cell: additional genes detected only in scRNA-seq; bulk: additional genes detected only in bulk RNA-seq. f, As in e for PTlower neurons. g, Total number of genes detected across all experiments. In e–g, single cell reads were downsampled such that total read depth was the same for scRNA-seq and bulk RNA-seq. h, Number of genes detected in scRNA-seq for each PTupper and PTlower neuron (‘X’, median; PTupper, 9,936 genes; PTlower, 9,865 genes). i, Mean fold change in expression (measured as log2(CPM + 1)) of all genes detected by both methods between neurons in the Npsr1–Hpgd clusters (PTupper) and neurons in the Slco2a1 cluster (PTlower) as determined by scRNA-seq (x axis) and bulk RNA-seq (y axis). Colour represents classification accuracy between the Npsr1–Hpgd and Slco2a1 clusters using a binary (detected/not detected) version of the scRNA-seq data. The 100 most discriminative neurons are coloured for each type. The correlation coefficient (Pearson’s r) in fold change expression was 0.87 for this set of 200 differentially expressed genes.
