Harmonized cross-species cell atlases of trigeminal and dorsal root ganglia

Peripheral sensory neurons in the dorsal root ganglion (DRG) and trigeminal ganglion (TG) are specialized to detect and transduce diverse environmental stimuli including touch, temperature, and pain to the central nervous system. Recent advances in single-cell RNA-sequencing (scRNA-seq) have provided new insights into the diversity of sensory ganglia cell types in rodents, non-human primates, and humans, but it remains difficult to compare transcriptomically defined cell types across studies and species. Here, we built cross-species harmonized atlases of DRG and TG cell types that describe 18 neuronal and 11 non-neuronal cell types across 6 species and 31 studies. We then demonstrate the utility of this harmonized reference atlas by using it to annotate newly profiled DRG nuclei/cells from both human and the highly regenerative axolotl. We observe that the transcriptomic profiles of sensory neuron subtypes are broadly similar across vertebrates, but the expression of functionally important neuropeptides and channels can vary notably. The new resources and data presented here can guide future studies in comparative transcriptomics, simplify cell type nomenclature differences across studies, and help prioritize targets for future analgesic development. Beta-III tubulin:GAP43-EGFP transgenic axolotls (125) 7-8cm in length (snout to tail) were anesthetized in 0.1% tricaine. For DRG dissociation at single-cell level, 10X Genomics “Dissociation of Mouse Embryonic Neural Tissue for Single Cell RNA Sequencing” protocol was adapted to axolotl tissue. DRGs of brachial plexus nerves located at C3, C4 and C5 were collected from both left and right sides and combined (total of 6 DRGs) in a single tube containing chilled 0.7X HBSS buffer and kept on ice. For dissociation, 400ul papain (Worthington Biochemical, Cat: #LK003178) dissolved in 0.7X PBS was pre-warmed in 37˚C water bath for 10 minutes to activate the enzyme. HBSS was removed and activated papain was added onto DRGs. The DRG in papain was placed in 37˚C water bath for 20 minutes. After the incubation, DRGs were settled in the bottom of the tube by a brief spin at 200g for 1min and papain was removed. 500ul DRG media consisting of Neurobasal Plus Medium at 60% (v/v), (Thermo Scientific, cat: A3582901), 50X B-27 Plus Supplement stock at 1X (Thermo Scientific, cat: A3582801), 100X insulin-transferrin-selenium stock at 1% (v/v), (Peprotech, cat: 41400045), 250μg/mL amphotericin B stock at 1% (v/v), (Sigma-Aldrich, cat: A2942), gentamicin at 50mg/ml (Sigma-Aldrich, cat: G1264), 50ug/ml recombinant human β-NGF stock at 1:1000, (Peprotech, cat: 450-01) mixed in Ringer’s solution (115mM NaCl, 2.5mM KCl, 2mM CaCl2, 10mM HEPES pH7.4, 0.5 mM EDTA dissolved in water and ph adjusted to 7.4 with NaOH) was added. DRGs were triturated in media using 1000 μl pipette. Trituration was performed slowly and gently until most of the tissue was dissociated. After dissociation, sample was centrifuged at 200g for 3min. The supernatant was removed, and the pellet was resuspended in 100 μl of0.7XPBS+%0.04 BSA. Cell count and single-cell sequencing were performed by Harvard University Bauer Sequencing core. Luna-FL Dual Fluorescence Cell Counter was used to assess number of viable cells. Cell count was repeated 4 times and averaged to determine the final cell count, viability, and cell size. Total of 405 cells/μl with 100% cell viability were present in the final cell suspension in first sample and 486 cells/μl with 97% viability in the second set. scRNA-seq libraries were prepared with the Chromium™ Single Cell 3′ NextGEM, Library and Gel Bead Kit v3.1.