Fig. 2. Single cell transcriptome analysis of PV⁺Rx3⁺ neurons.

a Schematic of single cell RNA sequencing strategy employed in experiments. DRG from adult (≥p56) PVRx3:tdT mice were collected and dissociated into single cell suspensions. tdT⁺ SNs were isolated using FACS. To ensure cell viability, neurons were also assessed for the presence of high Calcein Blue (CalB) and absence of Sytox red (SytR) labeling while sorting individual neurons into a single well of a 96-well plate containing lysis buffer (see “Methods” for details). b t-stochastic neighbor embedding (tSNE) plot of adult cells, colored by cluster membership. Clustering was done using a gene module and bootstrapping approach (with the hicat package—see “Methods”). c–f tSNE plots of cells colored by expression of four genes. In each plot, the color represents no expression (grey) to maximum counts per million (CPM) value over all the cells (red). g Barplots showing a subset of differentially expressed genes across the five main clusters. For each gene, the maximum CPM value over all cells is shown on the right side of the corresponding bar plot. Genes were selected for differential expression across multiple pairs of clusters with the added restriction of binary (on-off) expression, as far as possible, in at least two of the clusters. h Expression of Heg1 and Pcdh8 in tdT⁺ pSNs in lumbar DRG of adult (≥p56) PVRx3:tdT mice. Single neuron analysis (i–iii) of Heg1 (h) and Pcdh8 (p) expression exhibits three expression patterns across the five molecular identified clusters: cluster 1 tdT⁺ neurons expressing high transcript levels of Heg1 but little Pcdh8 (i), cluster 2-4 tdT⁺ neurons with comparable levels of Heg1 and Pcdh8 (ii), and cluster 5 tdT⁺ neurons with high Pcdh8 but no Heg1 (iii). Some Pcdh8 expressing neurons fall outside of the tdT⁺ pSN population (marked by asterisk). i Expression of Nxph1 and Pcdh8 in tdT⁺ pSNs in lumbar DRG of adult PVRx3:tdT mice. Single neuron analysis (i–iii) of Nxph1 (n) and Pcdh8 (p) expression shows presumed (i) cluster 1 tdT⁺ neurons expressing no Nxph1 but low levels of Pcdh8 transcript, (ii) cluster 2–4 tdT⁺ neurons coexpressing Nxph1 and Pcdh8 (with variable levels of Pcdh8), and (iii) cluster 5 tdT⁺ neurons with no Nxph1 but high Pcdh8 transcript levels. Similar to Pcdh8, Nxph1 is also expressed in a few non-tdT⁺ neurons (marked by asterisk). j Percentage of Heg1⁺, Nxph1⁺, and Pcdh8⁺ tdT⁺ SNs in ≥p56 lumbar DRG of PVRx3:tdT animals. Data points indicate counts for a single DRG section and include both low and high expressing neurons. Mean percentage ± S.E.M. for Heg1: 63.5 ± 3.2%, n = 29 sections; for Nxph1: 57.8 ± 2.7%, n = 39 sections; for Pcdh8: 53.9 ± 1.9%, n = 94 sections. Similar data obtained from at least three (h, i) biological replicates. In boxplot (j), boxes indicate medians and 25th/75th percentiles, and whiskers extend to the furthest point less than 1.5 standard deviations from the mean. Scale 10 μm.