Single‐Cell <scp>RNA</scp> ‐Sequencing of Zebrafish Olfactory Epithelium Identifies Odor‐Responsive Candidate Olfactory Receptors

Single-cell RNA-sequencing (scRNA-seq) is a powerful method to comprehensively overlook gene expression profiles of individual cells in various tissues, providing fundamental datasets for classification of cell types and further functional analyses. Here we adopted scRNA-seq analysis for the zebrafish olfactory sensory neurons which respond to water-borne odorants and pheromones to elicit various behaviors crucial for survival and species preservation. Firstly, a single-cell dissociation procedure of the zebrafish olfactory rosettes was optimized by using cold-active protease, minimizing artifactual neuronal activation. Secondly, various cell types were classified into distinct clusters, based on the expressions of well-defined marker genes. Notably, we validated non-overlapping expressions of different families of olfactory receptors among the clusters of olfactory sensory neurons. Lastly, we succeeded in estimating candidate olfactory receptors responding to a particular odor stimulus by carefully scrutinizing correlated expressions of immediate early genes. Thus, scRNA-seq is a useful measure for the analysis of olfactory sensory neurons not only in classifying functional cell types but also in identifying olfactory receptor genes for given odorants and pheromones.