Single-cell transcriptomics of vomeronasal neuroepithelium reveals a differential endoplasmic reticulum environment amongst neuronal subtypes
- Tata Institute of Fundamental Research, India
- Department of Cell Biology, University of Connecticut Health Center, United States
Figures
Figure 1 with 1 supplement
Single-cell RNA sequencing of the mouse vomeronasal neuroepithelium.
(A) Uniform Manifold Approximation and Projection (UMAP) of 9185 cells from vomeronasal neuroepithelium with 18 identified clusters. Each point represents a cell that is color-coded according to the cell type. Clusters were assigned a cell type identity based on previously known markers. Percentage of total cells corresponding to each cluster is in Supplementary file 2. (B) Dot plot showing the expression of the top 10 gene markers for each cluster identified by differential expression analysis. Gene expression markers shared across multiple clusters are listed only once. The dot radius and dot color indicate the percentage of cells expressing the gene and scaled expression value in that cluster, respectively. Top 20 gene markers for each cluster with log2 fold change are listed in Supplementary file 1. (C) Feature plot showing the expression level of major known gene markers of neuronal and sustentacular cell types. Figure 1—figure supplement 1 shows UMAP clusters and gene expression comparison between males and females.
Figure 1—figure supplement 1
Comparison of cell type composition and gene expression from the male and female vomeronasal neuroepithelium.
(A) Uniform Manifold Approximation Projection (UMAP) of cells from male and female vomeronasal neuroepithelium, with the cluster numbers corresponding to Figure 1A and B. Solitary chemosensory neurons (Cluster 18) were seen only in female data. (B) Scatter plots comparing the average expression of genes across each cluster from male and female with Pearson correlation coefficient at the top of the plot. Each point in the plot represents a gene. Known sexually dimorphic genes: Eif2s3y, Ddx3y, Uty, and Kdm5d are marked in red. Scatter plot of cluster 17 between male and female is not shown due to low cell count. Differential gene expression between each cluster of males and females organized in a cluster-wise manner is shown in Figure 1—figure supplement 1—source data 1.
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Figure 1—figure supplement 1—source data 1
Average log2 fold change gene expression between male and female along with the fraction of cells expressing the gene in male or female cluster.
The comparison of each cluster is separated in sheets.
- https://cdn.elifesciences.org/articles/98250/elife-98250-fig1-figsupp1-data1-v1.xlsx
Figure 2
Gene markers of non-sensory cells in the vomeronasal organ identified from single-cell RNA sequencing.
(A) Uniform Manifold Approximation and Projection (UMAP) feature plot (top) of a single representative gene marker for non-sensory cell clusters and their spatial gene expression pattern by RNA in situ hybridization on Vomeronasal organ (VNO) coronal sections (bottom, scale bar: 100 µm). (B, C) Three clusters of macrophage-like cell types represented by UMAP projection (B) and a dot plot (C) showing differentially expressed genes between these clusters. The dot radius indicates the percentage of cells expressing a gene, whose scaled expression level is represented by the intensity of color as per the scale. Additional markers of each non-sensory cell type are in Supplementary file 1 and differential expression between macrophage clusters is in Supplementary file 3.
Figure 3 with 2 supplements
Gene expression dynamics during development of sensory neurons.
(A) Uniform Manifold Approximation and Projection (UMAP) of neuronal cells with clusters (n1-n13 from Figure 3—figure supplement 1) represented in different colors. Sub-clusters within mature Gnai2, and Gnao1 neurons are merged. The line on the UMAP plot shows the pseudotime developmental trajectory of neurons. (B) Volcano pot of differential gene expression between immature Gnai2 (cluster n6; Gap43+, Gnai2+) vs immature Gnao1 (cluster n7; Gap43+, Gnao1+) neurons. Genes that satisfy |log2 fold change normalized expression|>1 (green) and -log10 p-value >6 (blue) are considered significant and colored in red. Non-significant (NS) genes are colored in gray. Arrows point to transcription regulators enriched in Gnao1 + immature neurons. Complete list of differentially expressed genes is in Figure 3—source data 1. (C) Feature plots showing the normalized expression levels of previously known markers for immature neurons (Gap43), Gnao1 neurons (Tfap2e), Gnai2 neurons (Meis2), and transcription factor or related genes: Creb5, Prrxl1, Shisa8, Lmo4, Foxo1. Arrows highlight the limited expression of Creb5 and Prrxl1 to immature neurons, but are absent from mature indicating transient expression during the development of Gnao1 neurons. Subclusters within Gnao1, Gnai2 neurons and the effect of VRs on sub-clustering are in Figure 3—figure supplements 1 and 2 respectively.
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Figure 3—source data 1
Differentially expressed genes between immature Gnao1 and immature Gnai2 neurons.
Genes that satisfy the criteria |log2 foldchange|>1 and adjusted p-value <10–6 are saved in a separate sheet.
- https://cdn.elifesciences.org/articles/98250/elife-98250-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Neuronal cell types of vomeronasal sensory epithelium.
(A) Uniform Manifold Approximation and Projection (UMAP) projection of neurons with 13 clusters (n1–n13). (B) Feature plot showing expression of neuronal markers associated with various stages of differentiation: Globose basal cells (n5; Ascl1), progenitor cells (n5; Neurod1, Neurog1), immature neurons (n6, n7; Gap43+), mature Gnao1 neurons (n1-n4; Gap43-, Omp+, Gnao1+) and mature Gnai2 neurons (n8-n13; Gap43-, Gnai2+, Omp+). Arrows highlight the expression of Gnao1 and Gnai2 in Gap43+immature neurons (n6, n7). (C) Dot plot showing enriched genes in each cluster compared to all other clusters obtained by differential expression analysis. Size of the dot represents the percentage of cells expressing the gene in that cluster and the color indicates the scaled expression value. Top five gene markers based on log2fold change from each cluster were chosen by filtering the genes whose adjusted p-value is less than 0.005, expression in at atleast 50% of cells, and less than 50% of cells of all other clusters. No markers were found for cluster n9 with this filtering criteria. Complete list of the top 30 markers for neuronal clusters is in Supplementary file 4.
Figure 3—figure supplement 2
Effect of VR genes on neuronal clustering.
(A) Clustering of neurons based on the top 2000 variable geneset in the dataset leading to 13 clusters (same as Figure 3—figure supplement 1A). (B) Reclustering performed after excluding genes coding vomeronasal receptors from the variable geneset without changing any other parameters. The bifurcation of Gnao1, Gnai2 neurons at mature and immature stages remains unchanged. The only changes are the merger of mature Gnao1 sub-clusters n1/n3 and the change in mature Gnai2 n8 composition.
Figure 4 with 4 supplements
Subpopulation of Gnao1 neurons defined by vomeronasal type-2 GPCR (V2R) and H2-Mv expression.
(A) Uniform Manifold Approximation and Projection (UMAP) representation of Gnao1 neurons from Figure 3. Each dot represents a cell and four Gnao1 neuron clusters (n1–n4) are marked in different colors. (B–D) Feature plot showing exclusive expression of Vmn2r1 (B), Vmn2r2 (C), and the most abundant H2-Mv gene, H2-M10.3 (D) in Gnao1 neurons. (E) Heat map showing the expression of V2R and H2-Mv genes in Gnao1 neurons. Cluster numbers are marked on the top with color coding as in (A) and gene families are labeled on the left. Each column represents a cell and the scaled gene expression in each row is color coded as per the scale with red and blue indicating high and low expression, respectively. Vmn2r1 is expressed in almost all cells of cluster-1 and cluster-4; Vmn2r2 is expressed in all cells of Cluster 3; Cluster2 has mutually exclusive expression of Vmn2r1 and Vmn2r2. (F–H) Bar plot showing a number of cells expressing: 0–7 family-C vomeronasal type-2 GPCRs (V2Rs) per cell (F), 0–5 family-ABD V2Rs per cell (G), 0–6 H2-Mv genes per cell (H) with composition of cells associated with family C1 (orange) or C2 (blue) V2R color coded on the bar. (I) Box plots comparing the sum of normalized expression levels of family-C V2Rs and Gnao1 (Green) in a cell that expresses 1–5 family-C V2Rs. (J) Box plot comparing the level of total ABD-V2R expression from cells with 1 and 2 ABD-VRs along with Gnao1 expression level (green). (K) Box plot comparing the level of total H2-Mv expression in cells that express 1–5 H2-Mv genes along with Gnao1 expression level (Green). Multiple combinations of family-C, family ABD V2Rs, and H2-Mvs identified to be co-expressed in a single-cell and their cell frequency are listed in Supplementary file 5.
Figure 4—figure supplement 1
Frequency and distribution of vomeronasal type-2 GPCR (V2R) and H2-Mv genes.
Bar plot showing the number of cells expressing top 20 vomeronasal type-I GPCRs (V1Rs) (A), vomeronasal type-2 GPCRs (V2Rs) (B), and H2-Mvs (C). Normalized gene expression values were extracted for all V1Rs (D) from Gnai2 neurons, V2Rs (E), and H2-Mvs (F) from Gnao1 neurons and density was plotted to identify the distribution of gene expression. The red dashed line represents a normalized gene expression value that was used as the threshold to call the expression of respective genes.
Figure 4—figure supplement 2
Characteristics of H2-Mv expression.
(A, B) Feature plot showing the expression H2-M10 family genes (A) and limited expression of phylogenetically divergent H2-Mv members H2-M1, H2-M9, and H2-M11 (B) in Gnao1 neurons. (C) Feature plot showing the expression of Vmn2r81 and Vmn2r82 in a few cells of clusters 2 and 4 of Gnao1 neurons that express H2-M1, M9, and M11. (D) Scatter plot showing the normalized expression level per cell of rarely expressed H2-Mv genes (H2– M1, M9, and M11) on the x-axis and Vmn2r1 or Vmn2r2 on the y-axis indicating that H2-M1, M9 and M11 co-express with Vmn2r1, unlike other H2-Mv genes. (E) Two-color RNA in situ hybridization of H2-M1, H2-M9, and H2-M11 with Vmn2r81/82 confirming the co-expression. The in situ hybridization (ISH) probe was common for Vmn2r81 and 82; H2-M1/M9/M11 indicates pooling of individual probes. Scale bar: 100 μm.
Figure 4—figure supplement 3
Co-expression characteristics of vomeronasal type-2 GPCR (V2R) and H2-Mv genes using data from Hills et al., 2024.
(A–C) Bar plot showing the number of cells expressing: 0–7 family-C V2Rs per cell (A), 0–5 family-ABD V2Rs per cell (B), 0–6 H2-Mv genes per cell (C) with composition of cells associated with family C1 (orange) or C2 (blue) V2R color coded on the bar. The trend of co-expression is similar to Figure 7F, G and H indicating that co-expression counts are similar across datasets.
Figure 4—figure supplement 4
Co-expression of vomeronasal type-I GPCRs (V1Rs) in Gnai2 neurons.
(A) Heatmap showing the expression of selected V1Rs in Gnai2 neurons. Each column represents a cell, and the scaled expression value of each VR is colorcoded in each row with red and blue indicating high and low expression, respectively. A continuous red line in two rows of a single column indicates the expression of two receptors in a single-cell. (B) Bar plot showing the number of cells expressing 0–3 V1Rs per cell indicating the vomeronasal type-I GPCR (V1R) co-expression is limited to a small subset of cells. (C) Box plot comparing total V1R and Gnai2 (green) expression from cells shown in (B). Combinations of V1Rs co-expressed in a single-cell and their cell frequency are listed in Supplementary file 6.
Figure 5 with 1 supplement
Divergent gene expression: Gnao1 vs Gnai2 neurons.
(A) Volcano plot showing differentially expressed genes between mature Gnao1 and Gnai2 neurons. Genes that satisfy |log2 fold change normalized expression|>2 (green) and -log10 p-value >6 (blue) are considered significant and colored in red. Non-significant (NS) genes are colored in gray. Complete list of differentially expressed genes is in Figure 5—source data 1. (C–D) Two color RNA in situ hybridization (ISH) of selected enriched genes marked in bold on the volcano plot. Gnao1, Gnai2, respective markers of basal and apical neurons are shown in (B). Genes enriched in Gnai2 neurons (C) or Gnao1 neurons (D) are co-localized with the respective markers. RNA-ISH for additional enriched genes is shown in Figure 5—figure supplement 1. Scale bar:100 µm.
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Figure 5—source data 1
Differentially expressed genes between mature Gnao1 and mature Gnai2 neurons.
Genes that satisfy the criteria |log2 foldchange|>1 and adjusted p-value <10–6 are saved in a separate sheet.
- https://cdn.elifesciences.org/articles/98250/elife-98250-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Expression pattern of enriched genes in Gnai2 or Gnao1 neurons.
Chromogenic RNA-in situ hybridization (ISH) showing expression of Gnai2 enriched genes: Nsg1, Rtp1, Dner, Qpct, Pcdh7, Prph (A) and Gnao1 enriched genes: Apmap, Selenof, Hspa5 (Bip), Itm2b, Agpat5, Sncg (B). Sncg and Prph are expressed in a scattered pattern amongst a few neurons. Scale bar: 100 μm.
Figure 6
Enrichment of endoplasmic reticulum (ER) genes in Gnao1 neurons.
(A) ER gene expression in Gnao1 neurons. Annotation of gene ontology (GO) biological processes of genes that are significantly enriched in Gnao1 neurons from Figure 5A. GO terms related to ER processes are marked in red. p-value <0.05 was used as a cut-off. (B) Vomeronasal organ (VNO) coronal section two-color fluorescent RNA-in situ hybridization (ISH) of selected Gnao1 enriched ER chaperone genes (Creld2, Pdia6, Dnajc3, Sdf2l1: green), co-labeled with Gnao1 probe (red) shows Gnao1 zone restricted expression of these genes. Scale bar 100 µm.
Figure 7 with 2 supplements
Differential localization of endoplasmic reticulum (ER) proteins in Vomeronasal organ (VNO) neurons.
Pseudocolored immunofluorescence images of VNO coronal sections labeled with antibodies against KDEL (anti-SEKDEL), a common ER retention signal (A). The section is co-labeled with anti-Gnao1 to mark basal zone neurons (B) and anti-OMP to mark all mature neurons (C). Signal intensity of KDEL, Gnao1, and OMP channels are quantified from ROIs along the apical-basal axis as shown in example (D). Signal intensity measured from multiple sections (n>20 for each antibody) from three biological replicates was normalized and the trendline was fitted to show the Gnao1 neuron-enriched localization of anti-KDEL (E). Points on the plot show normalized intensity values color-coded for each antibody on which the trendline was fitted. Similar immuno-labeling and quantification of ER chaperone Hspa5 (BiP) (F), ER membrane translocon subunit Sec61β (G), and ER membrane protein Atlastin1 (H) indicate their enrichment in Gnao1 neurons compared to Gnai2 neurons. Distribution of additional ER chaperone and membrane proteins is shown in Figure 7—figure supplement 1 along with combined source data in Figure 7—source data 1. Scale bar: 50 µm.
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Figure 7—source data 1
Fluorescence intensity values of ROIs along an apical-basal axis from immunolabeled images of Vomeronasal organ (VNO) neuroepithelium.
- https://cdn.elifesciences.org/articles/98250/elife-98250-fig7-data1-v1.zip
Figure 7—figure supplement 1
Pseudocolored immunofluorescence images of Vomeronasal organ (VNO) coronal sections labeled with antibodies against endoplasmic reticulum (ER) proteins.
(A) Chaperone proteins: PDI/Grp94/Calnexin, (B) ER structural proteins: NogoB/Climp64/Reep5. Higher intensity in Gnao1 neurons compared to Gnai2 neurons is seen from the images and quantified along the apical-basal axis by co-labeling sections with anti-OMP to mark all neurons and anti-Gnao1 (or anti-SEKDEL -see Figure 7A–E depending on antibody species), to mark Gnao1 neurons. Fluorescence intensity along the apical-basal axis is quantified from at least 20 sections from three biological replicates. The normalized fluorescence intensity of ER proteins increases along the apical-basal axis, similar to Gnao1 or SEKDEL, while the level of OMP either remains the same or slightly decreases. Scale bar: 50 µm.
Figure 7—figure supplement 2
Comparison of endoplasmic reticulum (ER) gene expression between Gnai2, Gnao1 neurons.
Violin plots showing the gene expression levels in mature Gnao1 or Gnai2 neurons whose protein levels are shown via immunofluorescence in Figure 7 and Figure 7—figure supplement 1. Log2 fold change value for Gnao1 vs Gnai2 calculated from pseudobulk differential gene expression analysis is mentioned on top of the line and the Bonferroni-adjusted p-value is mentioned below the line (ns - not significant). RNA levels of Hspa5, Calnexin, Hsp90b1, Sec61b, and Reep5 are significantly upregulated in Gnao1 neurons while PDI and Atlastin1 do not differ significantly between Gnao1 and Gnai2 neurons.
Figure 8 with 1 supplement
Basal/Gnao1 neurons are densely packed with cubic membrane endoplasmic reticulum (ER).
(A) Scanning electron micrographs of the vomeronasal sensory epithelium at low magnification showing cell bodies of VNO sensory neurons (VSNs), sustentacular cells, and basal lamina. Boxed regions in red or green mark cell bodies of basal/Gnao1 or apical/Gnai2 neurons, respectively, that are displayed at higher magnification below. Nucleus (N) appears light and ER is dark. Cell bodies of basal/Gnao1 neurons are larger and occupied by a substantial amount of ER, in comparison to apical/Gnai2 neurons. (B, B’) Magnified micrographs show the cell body of a basal/Gnao1 neuron, packed with cubic ER membranes. White arrows point to dense membrane stacks that are better resolved in Figure 8—figure supplement 1. Red arrow points to the lamellar ER membrane that is contiguous with the cubic membrane. (C, C’) Cell bodies of apical/Gnai2 neurons also seem to show dense cubic membrane ER, that is smaller in comparison to basal/Gnao1 neurons.
Figure 8—figure supplement 1
Cubic membrane endoplasmic reticulum (ER) in Gnao1 neurons.
Scanning electron micrographs showing cell bodies of basal/Gnao1 neurons with sinusoidal ER membranes interspersed with stacked membranes (arrows).
Figure 9
Onset of vomeronasal type-2 GPCR (V2R) expression coincides with the expression of endoplasmic reticulum (ER) chaperone genes.
(A) Feature Plot showing the expression of Gnao1, Vmn2r1, Sdf1l1, and Manf. Sdf2l1 and Manf are known ER chaperones and their upregulation in Gnao1 neurons coincides with Vmn2r1 expression, which is preceded by Gnao1 expression. (B) Heatmap showing the expression of Gnao1, Vmn2r1, Vmn2r2, and several ER chaperone genes (red) in the clusters arranged as per their developmental trajectory. (C) Cartoon summarizing major transcription factor expression during development leading to Gnao1 neurons with chaperone-rich hypertrophic ER compared to Gnai2 neurons.
Author response image 1
Author response image 2
ROIs (yellow) are manually drawn in the sensory epithelium, wherever possible to identify ER without ambiguity.
Area and centroid of ROI are calculated and x coordinates of centroid of each ROI are used to position ER area along the apical-basal axis as shown in the plot below.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Other | Papain | Worthington Biochemical corporation | LK003178 | Enzyme used for single-cell dissociation |
| Other | RNAse-free Deoxyribonuclease I | Roche | 4716728001 | Enzyme used for single-cell dissociation |
| Chemical compound, drug | EDTA | Himedia | ML014-500ML | |
| Other | Ovomucoid inhibitor and albumin mix | Worthington Biochemical corporation | LK003182 | Used in single-cell dissociation |
| Chemical compound, drug | Glycerol | Sigma | G6279-1L | |
| Other | 2 mL Protein Lo-Bind tubes | Eppendorf | 022431102 | Used for single-cell dissociation |
| Other | Pasteur pipettes | Fisher | 13-678-8B | Used in single-cell dissociation |
| Other | HBSS | Hyclone | SH30268.02 | Used in single-cell dissociation |
| Other | EBSS | Hyclone | SH30029.02 | Used in single-cell dissociation |
| Other | 40 um cell strainer | Pluriselect | 43-10040-60 | Used in single-cell dissociation |
| Other | 70 um cell strainer | Pluriselect | 43-10070-60 | Used in single-cell dissociation |
| Commercial assay or kit | Chromium Next GEM Single-Cell 3' Kit v3.1, 4rxns | 10 X Genomics | 1000269 | |
| Commercial assay or kit | Chromium Next GEM Chip G Single-Cell Kit | 10 X Genomics | 1000120 | |
| Commercial assay or kit | GoTaq DNA polymerase | Promega | M3005 | |
| Commercial assay or kit | NucleoSpin Gel and PCR Clean-up, Mini kit | Macherey-Nagel | 740609.250 | |
| Antibody | Anti-Digoxigenin-AP (purified Fab fragments from Sheep) | Roche | Cat#11093274910 RRID: AB_514497 | Dilution - 1:7500 |
| Antibody | Anti-Digoxigenin-POD (purified Fab fragments from Sheep) | Roche | Cat#11207733910 RRID: AB_514500 | Dilution - 1:500 |
| Antibody | Anti-Fluorescein-AP (purified Fab fragments from Sheep) | Roche | Cat#11426338910 RRID: AB_2734723 | Dilution - 1:7500 |
| Antibody | Anti-Fluorescein-POD (purified Fab fragments from Sheep) | Roche | Cat#11426346910 RRID: AB_840257 | Dilution - 1:500 |
| Commercial assay or kit | 10 X Flu RNA labeling mix | Roche | 11685619910 | |
| Commercial assay or kit | 10 X Dig RNA labeling mix | Roche | 11277073910 | |
| Peptide, recombinant protein | T7 RNA polymerase | Promega | P2075 | |
| Peptide, recombinant protein | SP6 RNA polymerase | Promega | P1085 | |
| Chemical compound, drug | 5 X Transcription optimized buffer | Promega | P1181 | |
| Peptide, recombinant protein | RNasin Plus | Promega | N2611 | |
| Chemical compound, drug | 100 mM DTT | Promega | P1171 | |
| Chemical compound, drug | Diethyl pyrocarbonate | Sigma | 40718–25 ML | |
| Chemical compound, drug | Akoya Blocking powder | Akoya | FP1020 | |
| Chemical compound, drug | TSA plus Cy3 | Akoya | NEL744001KT | |
| Chemical compound, drug | TSA Plus Fluorescein | Akoya | NEL741001KT | |
| Commercial assay or kit | BCIP/NBT kit | Promega | S3771 | |
| Other | Bovine Serum Albumin | Jackson ImmunoResearch Inc | Cat#001-000-173 RRID:AB_2336947 | Blocking reagent in IHC |
| Other | OCT freezing medium | Leica | Cat#14020108926 | Used for freezing tissue |
| Chemical compound, drug | Paraformaldehyde | Electron Microscopy Sciences | 157–8 | |
| Antibody | anti-Omp (Goat polyclonal) | Wako | Cat#019–22291 RRID:AB_3094987 | Dilution –1:2000 |
| Antibody | anti-Gnao1/GαO (Rabbit polyclonal) | MBL life science | Cat#551 RRID:AB_591430 | Dilution –1:2000 |
| Antibody | anti-SEKDEL ER marker (Mouse monoclonal) | Santa Cruz | Cat #sc-58774 RRID:AB_784161 | Dilution –1:1000 |
| Antibody | anti-Grp94 (Rat monoclonal) | Invitrogen | Cat#MA3-016 RRID:AB_2248666 | Dilution –1:1000 |
| Antibody | anti-Hspa5/BiP (Mouse monoclonal) | BD | Cat#610978 RRID:AB_398291 | Dilution –1:1000 |
| Antibody | anti-Atlastin1 (Rabbit polyclonal) | Invitrogen | Cat#PA5-85682 RRID:AB_2792821 | Dilution –1:500 |
| Antibody | anti-PDI (Mouse monoclonal) | Enzo | Cat#ADI-SPA-891 RRID:AB_10615355 | Dilution –1:500 |
| Antibody | anti-Sec61β (Rabbit polyclonal) | Invitrogen | Cat#PA3-015 RRID:AB_2239072 | Dilution –1:400 |
| Antibody | anti-Calnexin (Rabbit polyclonal) | Abcam | Cat#ab10286 RRID:AB_2069009 | Dilution –1:500 |
| Antibody | anti-Reep5 (Rabbit polyclonal) | ProteinTech | Cat#14643–1-AP RRID:AB_2178440 | Dilution –1:500 |
| Antibody | anti-NogoB (Rabbit recombinant monoclonal) | Invitrogen | Cat#MA5-32763 RRID:AB_2810040 | Dilution –1:500 |
| Antibody | anti-Ckap4 (Rabbit polyclonal) | ProteinTech | Cat#16686–1-AP RRID:AB_2276275 | Dilution –1:500 |
| Antibody | anti-Mouse IgG- Alexa Fluor 647 (Donkey polyclonal) | Jackson ImmunoResearch Inc | Cat#715-605-150 RRID:AB_2340862 | Dilution –1:1600 |
| Antibody | Anti-Rat IgG-Cy3 (Donkey polyclonal) | Jackson ImmunoResearch Inc | Cat#712-165-153 RRID:AB_2340667 | Dilution –1:1600 |
| Antibody | Anti-Rabbit IgG-Cy3 (Donkey polyclonal) | Jackson ImmunoResearch Inc | Cat#711-165-152 RRID:AB_2307443 | Dilution –1:1600 |
| Antibody | Anti-Goat IgG-Alexa Fluor 488 (Bovine polyclonal) | Jackson ImmunoResearch Inc | Cat#805-545-180 RRID:AB_2340883 | Dilution –1:1600 |
| Software, algorithm | Cell Ranger v5.0.1 | 10 x genomics; Zheng et al., 2017 | RRID:SCR_023221 | |
| Software, algorithm | Seurat v4.3.0 | Satija Lab; Hao et al., 2021 | RRID:SCR_016341 | |
| Software, algorithm | SoupX v1.6.2 | Young and Behjati, 2020 | RRID:SCR_019193 | https://github.com/constantAmateur/SoupX |
| Software, algorithm | TrimGalore v0.6.6 | TrimGalore | RRID:SCR_011847 | https://github.com/FelixKrueger/TrimGalore/releases/tag/0.6.6 |
| Software, algorithm | Clusterprofiler v4.2.2 | Wu et al., 2021 | RRID:SCR_016884 | https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html |
| Software, algorithm | Slingshot v2.2.1 | Street et al., 2018 | RRID:SCR_017012 | https://bioconductor.org/packages/release/bioc/html/slingshot.html |
| Software, algorithm | EnchancedVolcano v1.12.0 | EnhancedVolcano | RRID:SCR_018931 | https://github.com/kevinblighe/EnhancedVolcano |
| Software, algorithm | ggplot2 v3.5.0 | ggplot2 | RRID:SCR_014601 | https://ggplot2.tidyverse.org |
| Software, algorithm | Adobe illustrator CC | Adobe | https://www.adobe.com/products/illustrator | |
| Software, algorithm | ShinyCell | Ouyang, 2021 | RRID:SCR_022756 | https://github.com/SGDDNB/ShinyCell |
Additional files
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Supplementary file 1
List of top 20 genes specific to each cluster corresponding to Figure 1A and B.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp1-v1.xlsx
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Supplementary file 2
Frequency of cells belonging to various clusters corresponding to Figure 1A.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp2-v1.xlsx
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Supplementary file 3
List of top 30 highly expressed genes in three macrophage clusters (corresponding to Figure 2B and C) selected based on log2Fold change comparison of each cluster with the other two clusters.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp3-v1.xlsx
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Supplementary file 4
A list of the top 30 markers of neuronal clusters (n1-n13) from Figure 3—figure supplement 1, identified based on average log2FC after applying filters: adjusted p-value <0.005, fraction of cells expressing the gene in the cluster >=0.5 and fraction of cells expressing the gene other clusters <0.5.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp4-v1.xlsx
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Supplementary file 5
Frequency of co-expression of vomeronasal type-2 GPCRs (V2Rs) and H2-Mv genes in Gnao1 neurons (corresponding to Figure 4).
The frequency of family C, ABD V2Rs, H2-Mv, V2Rs-H2-Mv co-expression combinations are separated into sheets.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp5-v1.xlsx
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Supplementary file 6
Frequency of expression of vomeronasal type-I GPCRs (V1R) combinations in mature Gnai2 neurons, corresponding to Figure 4—figure supplement 4.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp6-v1.xlsx
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Supplementary file 7
Gene ontology terms (biological processes category) associated with genes for which RNA in situ hybridization was performed in Figure 6 and Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp7-v1.xlsx
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Supplementary file 8
Primer sequences used for PCR amplification of gene-specific DNA template to generate riboprobes for in situ hybridization (ISH)col.
- https://cdn.elifesciences.org/articles/98250/elife-98250-supp8-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/98250/elife-98250-mdarchecklist1-v1.docx
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Single-cell transcriptomics of vomeronasal neuroepithelium reveals a differential endoplasmic reticulum environment amongst neuronal subtypes
eLife 13:RP98250.
https://doi.org/10.7554/eLife.98250.3
