key: cord-0989355-xi419of3 authors: Baxter, B. Dnate’; Larson, Eric D.; Feinstein, Paul; Polese, Arianna Gentile; Bubak, Andrew N.; Niemeyer, Christy S.; Merle, Laetitia; Shepherd, Doug; Ramakrishnan, Vijay R.; Nagel, Maria A.; Restrepo, Diego title: Transcriptional profiling reveals TRPM5-expressing cells involved in viral infection in the olfactory epithelium date: 2020-05-15 journal: bioRxiv DOI: 10.1101/2020.05.14.096016 sha: fd6a915f6b22ce8a1ef47f90dd3833edef82c308 doc_id: 989355 cord_uid: xi419of3 Understanding viral infection of the olfactory epithelium is essential because smell loss can occur with coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), and because the olfactory nerve is an important route of entry for viruses to the central nervous system. Specialized chemosensory epithelial cells that express the transient receptor potential cation channel subfamily M member 5 (TRPM5) are found throughout the airways and intestinal epithelium and are involved in responses to viral infection. Herein we performed deep transcriptional profiling of olfactory epithelial cells sorted by flow cytometry based on the expression of fluorescent protein markers for olfactory sensory neurons and TRPM5. We find profuse expression of transcripts involved in inflammation, immunity and viral infection in TRPM5-expressing microvillous cells and olfactory sensory neurons. These cells express the Tmprss2 transcript that encodes for a serine protease that primes the SARS-CoV-2 spike protein before entry into host cells. Our study provides new insights into a potential role for TRPM5-expressing cells in viral infection of the olfactory epithelium. We did not find major differences in transcriptome profiling between males and females for 202 genes that were differentially expressed between the three cell groups ( Figure 5 -figure 203 supplement 3,4). We found a substantial number of olfactory receptor genes that were 204 differentially expressed between males and females ( Inc. Frozen slides were allowed to thaw and dry, then immersed in 70% ethanol overnight at 554 4°C, and allowed to dry again completely. Slides were inverted and placed on a Plexiglas 555 platform inside a humidified chamber; subsequent steps were performed using this setup. Slides 556 were incubated in 10 µg/µl proteinase K for 10 minutes at 37°C, then pre-hybridized with HCR 557 hybridization buffer (30% formamide buffer from Molecular Instruments) for 10 minutes at 558 37°C. Trpm5-B3 probes and OMP-B2 probes (0.4 pmol of each probe in 100 µl HCR 559 hybridization buffer per slide) were added, and slides were hybridized overnight at 37°C. Slides 560 were briefly incubated in undiluted HCR Wash Buffer (30% formamide buffer from Molecular 561 Instruments) at 37°C. Excess probes were removed by incubating slides for 15 minutes each at 37°C in solutions of 75% HCR Wash Buffer / 25% SSCT (5X SSC, 0.1% Tween, diluted in 563 RNAse free water), 50% Buffer / 50% SSCT, 25% Buffer / 75% SSCT, and 100% SSCT. Slides 564 were incubated in Amplification Buffer (Molecular Instruments) at room temperature for 30 565 minutes. 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Magenta: mCherry, green: 908 eGFP. Scale bar: i-iii, 50 µm, iv For both a and b, row and column order were determined automatically by the pHeatmap Nagel 5 , and Diego Restrepo 1 Figure 6-figure supplement 5. Significant differences in inflammation gene ontology for 1046 Figure 6-figure supplement 6. Significant differences in immune gene ontology for 1050 Figure 6-figure supplement 7. Significant differences in immune gene ontology for 1056