Abstract
We describe an approach for the high-throughput surveying of odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) that have been activated by specific odorants. When OSNs are activated, there is a molecular signature in the form of a phosphorylated-S6 (pS6) ribosomal subunit. By the immunoprecipitation of the protein-RNA complex containing pS6, we identify the OR mRNA species expressed in these activated OSNs. The one neuron - one receptor rule (mature OSN expresses a single unique OR) allows for the identification of the collection of ORs that responded toward the tested odorant. Here we detail the procedure of (1) odor stimulation, (2) tissue harvesting, (3) immunoprecipitation, and (4) mRNA profiling for the high-throughput deorphanization of ORs in vivo.
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Acknowledgments
This work was supported by the National Institutes of Health (DC014423 and DC016224) and National Science Foundation (1556207). We thank Yue Jiang for establishing the initial version of the protocol and Kentato lkegami for providing support on the bioinformatics pipeline. Maira Nagai, Aashutosh Vihani, and Marcelo Zapata helped to read and comment on the chapter.
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Hu, X.S., Matsunami, H. (2018). High-Throughput Odorant Receptor Deorphanization Via Phospho-S6 Ribosomal Protein Immunoprecipitation and mRNA Profiling. In: Simoes de Souza, F., Antunes, G. (eds) Olfactory Receptors. Methods in Molecular Biology, vol 1820. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8609-5_8
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DOI: https://doi.org/10.1007/978-1-4939-8609-5_8
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