Single-nucleus transcriptomic analysis of human dorsal root ganglion neurons
- National Institute of Dental and Craniofacial Research, National Institutes of Health, United States
- Department of Anesthesiology, College of Medicine, University of Cincinnati, United States
Figures
Figure 1 with 5 supplements
Diverse classes of human dorsal root ganglia (DRG) neurons revealed by single nuclear transcriptomics.
(A) Universal manifold (UMAP) representation of graph-based co-clustered snRNA sequences from human DRG nuclei reveal two well separated groups corresponding to sensory neurons (colored) and …
Figure 1—figure supplement 1
Human dorsal root ganglia (DRG) neurons express NeuN.
(A) Confocal image showing section of a DRG ganglion stained using NeuN histochemistry (red) and neurotrace (green). Note that the large diameter neurons are strongly NeuN positive whereas …
Figure 1—figure supplement 2
Support for the clustering of human dorsal root ganglia (DRG) neurons.
(A) Universal manifold (UMAP) representation showing relative expression levels of the neuronal marker SNAP25 (blue) and the non-neuronal gene APOE (red) in the initial clustering of sn-RNA …
Figure 1—figure supplement 3
Robust clustering of human dorsal root ganglia (DRG) neurons.
Universal manifold (UMAP) representations showing analysis of human DRG neurons using different parameters in Seurat. The choice of principal components (PCs) used for UMAP display and clustering is …
Figure 1—figure supplement 4
Additional markers that support similarities between dorsal root ganglia (DRG) neuronal clusters across species.
Universal manifold (UMAP) representations: upper panels, human sn-RNA sequencing; lower panels, mouse sn-RNA sequencing. To the left, identity of the different neuronal types is differentially …
Figure 1—figure supplement 5
Dotplots of gene expression supporting similarity of several dorsal root ganglia (DRG) neuronal classes in mice and humans.
Dotplots displaying information about the fractional expression and relative expression level of marker genes in the different identity classes of mouse (left, gray-green scale) and human (right, …
Figure 2 with 2 supplements
Human DRG neurons exhibit specialization that distinguishes them from mouse counterparts.
(A) Universal manifold (UMAP) representation of mouse and human dorsal root ganglia (DRG) neurons showing relative expression level (blue) of two genes that have been linked to pain sensation in …
Figure 2—figure supplement 1
Quantification of in situ hybridization (ISH) data.
The extensive autofluorescence in ISH of human dorsal root ganglia (DRG) section prevents automated signal detection. Thus, quantitation of ISH data involves manually scoring positive and negative …
Figure 2—figure supplement 2
H10 and H11 are classes of human dorsal root ganglia (DRG) neurons that express a range of itch-related genes.
(A) Dotplots displaying information about the fractional expression and relative expression level of marker genes in the different identity classes of mouse (left, gray-green scale) and human …
Figure 3 with 1 supplement
Co-clustering of human and mouse neurons support tentative assignments based on select genes.
(A) Universal manifold (UMAP) representation of the co-clustering of mouse and human neurons. Upper panel shows the mouse neurons colored by their identity when analyzed alone (Figure 1—figure …
Figure 3—figure supplement 1
More detailed analysis of relationships between human and mouse cell types.
(A) Universal manifold (UMAP) representation of clustered mouse neurons using parameters that identify 19 different groups of cells and (B) their distribution in the co-clustering with human …
Figure 4 with 2 supplements
Transcriptomically related classes of human dorsal root ganglia (DRG) neurons are spatially clustered in the ganglion.
Confocal images of sections through a human DRG probed for expression of key markers using multiplexed in situ hybridization (ISH); see Figure 4—figure supplement 1 for the individual panels and …
Figure 4—figure supplement 1
Expression profiles of clusters of H8 (cool), H9 (human-specific), and H15 (proprioceptive) neurons.
Individual channels for the in situ hybridization (ISH) images in Figure 4 are shown to highlight the expression patterns described in the text. Strong autofluorescence signals that are present in …
Figure 4—figure supplement 2
Expression profiles of clusters of H8 (cool), H9 (human-specific), and H15 (proprioceptive) neurons.
(A) Confocal images of mouse lumbar dorsal root ganglia (DRG) in situ hybridization (ISH) showing Nefh (green) and Scn10a (red); scale bars = 100 µm. (B) The nearest n neighbors of Nefh and Scn10a …
Figure 5 with 2 supplements
Two related classes of human nonpeptidergic small diameter neurons that may mediate itch.
(A) Confocal image of a section through a human dorsal root ganglia (DRG) probed for nociception-related genes using multiplexed in situ hybridization (ISH). In this view, many neurons expressing …
Figure 5—figure supplement 1
Expression profiles of human H10 and H11 dorsal root ganglia (DRG) neurons.
(A) Confocal in situ hybridization (ISH) images illustrating weak expression of itch-related transcripts HRH1 (upper panels, red) and NPPB (lower panels, red) in H10 and H11 neurons. Left image …
Figure 5—figure supplement 2
Gene expression patterns in H10 and H11 classes of human dorsal root ganglia (DRG) neurons are distinct from classes of mouse small diameter nonpeptidergic neurons.
(A) Dotplots displaying information about the fractional expression and relative expression level of marker genes in the different identity classes of mouse (left, gray-green scale) and human …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6NCrl | Charles River | Strain code: 027 | Male and female |
| Antibody | anti-NeuN (rabbit polyclonal) | Millipore | Cat#ABN78 | (1:4000 for nuclei isolation)(1:1000 for IHC) |
| Antibody | anti-Tubb3 (mouse monoclonal) | Proteintech | Cat#66375-1-Ig | (1:500) |
| Antibody | anti-rabbit Cy3-conjugated(donkey polyclonal) | JacksonImmuno Research | Cat#711-166-152 | (1:1000) |
| Antibody | anti-mouse FITC-conjugated(donkey polyclonal) | JacksonImmuno Research | cat#715-096-150 | (1:1000) |
| Sequence-based reagent | Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3.1 | ×10 Genomics | Cat#1000128 | |
| Sequence-based reagent | Chromium Next GEM Chip G Single Cell Kit | ×10 Genomics | Cat#1000127 | |
| Commercial assay or kit | RNAscope HiPlex8 Detection reagents | Advanced Cell Diagnostics | Cat#324110 | |
| Commercial assay or kit | RNAscope HiPlex12 Ancillary reagents | Advanced Cell Diagnostics | Cat#324120 | |
| Commercial assay or kit | Human RNAscope probes (HiPlex 12) | Advanced Cell Diagnostics | NEFH (cat# 448141); TRPM8 (cat# 543121); PIEZO2 (cat# 449951); SCN10A (cat# 406291); NTRK2 (cat# 402621); TAC1 (cat# 310711); OSMR (cat# 537121); SST (cat# 310591); TRPV1 (cat# 415381); PVALB (cat# 422181) | |
| Commercial assay or kit | RNAscope Fluorescent Multiplex assay | Advanced Cell Diagnostics | Cat #320851 | |
| Commercial assay or kit | Mouse RNAscope probes(MultiPlex) | Advanced Cell Diagnostics | Scn10a (cat#426011); Nefh (cat#443671) | |
| Commercial assay or kit | Human RNAscope probes(MultiPlex) | Advanced Cell Diagnostics | OSMR (cat#537121); SST (cat# 310591); HRH1 (cat#416501); NPPB (cat#448511); | |
| Commercial assay or kit | NeuroTrace Green | Fisher Scientific | Cat#N21480 | (1:100) |
| Software, algorithm | CellRanger | ×10 Genomics | Version 2.1.1 | GRCh38.v25. premRNA |
| Software, algorithm | Seurat | Satija lab | Versions 3-4.04 | https://satijalab.org/seurat/ |
| Software, algorithm | Python | python.org | Version 3.7. | |
| Software, algorithm | scipy.stats | scipy.org | Version 1.5.2 | |
| Software, algorithm | scikit-learn | scikit-learn.org | Version 0.23.2 | |
| Software, algorithm | RStudio | https://www.rstudio.com/ | Version 1.4.1106 | |
| Software, algorithm | R | https://www.r-project.org/ | R version 4.1.1 | |
| Software, algorithm | DoubletFinder | https://github.com/chris-mcginnis-ucsf/DoubletFinder | McGinnis lab (McGinnis, 2021) | |
| Software, algorithm | ImageJ | http://imagej.nih.gov/ij | ImageJ 1.53 c | |
| Software, algorithm | Adobe Photoshop | https://www.adobe.com/ | 25.5.1 release | |
| Software, algorithm | kl_divergence | https://gist.github.com/lars-von-buchholtz/636f542ce8d93d5a14ae52a6c538ced5636f542ce8d93d5a14ae52a6c538ced5 | ||
| Other | RNA-later | Thermo Fisher | Cat# AM7021 | |
| Other | Spectrum Bessman tissue pulverizer | Fisher Scientific | Cat# 08-418-3 | |
| Other | Dounce homogenizer | Fisher Scientific | Cat# 357538 | |
| Other | 40 μm cell strainer | Thermo Fisher | Cat# 08-771-1 | |
| Other | Low bind microfuge tubes | Sorenson BioScience | Cat# 11,700 | |
| Other | SUPERaseIn RNase inhibitor | Thermo Fisher | Cat# AM2696 | 0.2 U/μl |
| Other | anti-rabbit IgG microbeads | Miltenyi biotec | Cat# 130-048-602 | |
| Other | LS column | Miltenyi biotec | Cat# 130-042-401 | |
| Other | Mouse DRG dataset | Woolf lab | GSE154659 | Renthal et al., 2020, Neuron |
Table 1
Sequencing data for the five individual DRG nuclear preparations.
| Preparation# | DRG1-F36 | DRG2-M36 | DRG3a-F34 | DRG3b-F34 | DRG4-F35 | DRG5-F55 |
|---|---|---|---|---|---|---|
| Number of reads | 273,123,313 | 121,333,026 | 105,113,253 | 101,427,234 | 90,306,793 | 94,069,716 |
| Valid barcodes | 97.50% | 97.60% | 98.20% | 96.10% | 93.60% | 97.90% |
| Sequencing saturation | 93.20% | 93.10% | 39.70% | 82.10% | 38.70% | 55.90% |
| Q30 bases in barcode | 96.30% | 97.40% | 97.80% | 97.50% | 97.70% | 96.80% |
| Q30 bases in RNA read | 88.30% | 85.90% | 87.40% | 91.50% | 93.40% | 92.20% |
| Q30 bases in sample index | 96.00% | 96.80% | 97.40% | 96.60% | 96.60% | 94.80% |
| Q30 bases in UMI | 95.50% | 97.00% | 97.60% | 97.00% | 97.20% | 96.10% |
| Reads mapped confidently to genome | 86.50% | 81.20% | 71.20% | 16.90% | 10.80% | 18.10% |
| Reads mapped confidently to intergenic regions | 4.10% | 4.20% | 4.00% | 1.30% | 0.90% | 1.20% |
| Estimated number of cells | 584 | 273 | 6,180 | 223 | 872 | 999 |
| Mean reads per cell | 467,676 | 444,443 | 17,008 | 454,830 | 135,189 | 94,163 |
| Median genes per cell | 1917 | 709 | 786 | 481 | 872 | 812 |
| Total genes detected | 24,646 | 17,559 | 28,759 | 17,338 | 24,798 | 25,027 |
| Median UMI counts per cell | 2929 | 862 | 965 | 663 | 1,293 | 988 |
| DRG cells in final object | 212 | 152 | 770 | 80 | 281 | 342 |
