Demographics of dogs for DRG sequencing.

Supplementary Table – Demographics of dogs for DRG sequencing.

Number Dog Breed Sex

Age (yrs)

Neuter status

R01 Rottweiler M 9 1

R02 Labrador Retriever F 8 0

R03 Greyhound M 8 1

R04 Great Dane F 8 1

R05 Labrador Retriever F 9 0

O 02 Mixed Breed F 13 1

O 03 Golden Retriever M 10 1

O 04 Mixed Breed F 9 1

O 05 Bullmastiff M 8 1

O 06 Labrador Retriever M 10 1

O 07 Labrador Retriever F 7 1

O 08 Leonberger M 8 1

*C 01 Cocker Spaniel M 15 0

C 02 Labrador Retriever M 12 1

C 03 Mixed breed M 6 1

C 04 Labrador Retriever M 12 1

C 05 Scottish Deerhound M 8 0

C 06 Weimaraner M 8 1

Supplementary Figure 1 - Identification and quantification of markers for the TRPV1+

DRG neuronal subset targeted by RTX. Six RNA-Seq datasets were analyzed in the present heatmap analysis. Within each of the first three datasets (columns 1-3), one group was compared to the other and the ratio of expression is plotted according to the scale (right). Rat trigeminal ganglia were treated in vitro with either RTX to kill TRPV1+ neurons, or DMSO (column 1). The genes most highly decreased by RTX treatment are shown, sorted by level of decrease. This list is filtered by several subsequent criteria. As sensory ganglia contain non-

neural cells, DRG was compared to sciatic nerve (mainly Schwann cells) to reveal the neural subset of genes (column 2) and only those genes enriched in DRG vs. sciatic nerve are shown.

TRPV1+ lineage neurons were selectively sorted from mouse ganglia and sequenced, and compared to mice in which all of the TRPV1 lineage neurons were genetically ablated (column 3). Genes enriched in the TRPV1 lineage are shown. Single cell sequencing data showing fraction of positive cells in each DRG subclass are plotted (column 4), with the TRPV1+

populations highlighted. The non-peptidergic 1 population does not express TRPV1 but also shows a similar gene expression pattern. Expression values for the whole dog DRG and values for the whole human DRG are shown to the right (sFPKM).

Oprm1

Ntrk1 Tac1

Calca Trpa1

Cacna2d1 P2rx3

Trpv1 Merge

13.7%

33.8%

38.7%

33%

18.6%

27.0%

32.8%

Scn9a

38.2%

Expression of transcript > 5 FPKM Expression of both transcript > 5 FPKM No expression of either transcript > 5 FPKM Expression of transcript > 5 FPKM

t SNE 1 t SNE 1

t SNE 2t SNE 2t SNE 2t SNE 2

Supplementary Figure 2 - Clustering of selected nociceptive DRG genes. Data were mined from a publicly available database published by Li et al.³⁰. Single cells in this database were visualized using the Barnes-Hut implementation of the t-Distributed Stochastic Neighbor Embedding (t-SNE) technique according to their gene expression profiles³². Cells with similar gene expression profiles are plotted near each other in the 2-dimensional embedding. Cells were colored based on the expression of select genes (indicated at the bottom right of each panel). As can be seen in the visualization, all of these genes have some degree of coexpression

with Trpv1 expressing neurons. The lower limit cutoff was set at >5 FPKM (see legend, bottom ). Plots were generated using the Rtsne package in R version 3.3.1.

Supplementary Figure 3. Relationship between analgesic targets among Trpv1 expressing dorsal root ganglion cells by single-cell RNA sequencing. Data is from publicly available database of mouse DRG published by Li et al. (2016). A. Coexpression of Trpv1 with known analgesic targets. Cells with expression >5 FPKM for Trpv1 are shown in red. Percent of Trpv1+

cells with expression >5 FPKM for each analgesic target is shown in the upper right of each panel, highlighting substantial overlap between cells that coexpress Trpv1 and other analgesic targets. B. Coexpression of known and potential analgesic targets among Trpv1+ cells. As RTX ablates Trpv1+ nerve terminals, it preempts the need for other peripherally acting analgesics that might have acted on these peripheral nociceptors. C. The percent of Trpv1+ cells that express both genes (with expression >5 FPKM) is expressed in the diagonal matrix; many Trpv1+ cells express multiple analgesic targets.

Supplementary Figure 4 – Expression of TRPV1 in human tissues relevant to arthritis.

RNA-Seq data were mined and analyzed using MAGIC, and are reported in significant fragments per kilobase per million aligned reads (sFPKM). A. These data show abundance of TRPV1 expression in several human tissues, with the highest level of expression in sensory ganglia, where the encoded channel transduces heat and other sensations at the nerve terminal ending. The expression of this channel is also detectable in several tissues such as skin, placenta, and in the joint condyle, where it is much lower. B. An inset using a different Y-axis shows that skin and placenta express higher levels of TRPV1 than condoyle or chondrocytes, although TRPV1 is detectable in all five tissues.