pS6 immunoprecipitation RNA-seq of mouse olfactory epithelium in response to single odorants, binary mixtures, and complex fragrances

Phosphorylation of ribosomal protein S6 (pS6) serves as a molecular marker of neuronal activation by external stimuli. In this study, olfactory epithelium tissues were collected from mice exposed to single odorants (acetophenone, decanal, octanal, and cis-3-hexenol), binary mixtures (acetophenone–decanal and octanal–cis-3-hexenol), or complex fragrances (floral, mint, and floral–mint combination). To selectively capture transcripts undergoing active translation in odor-activated cells, pS6-associated ribosome complexes were isolated from tissue lysates through immunoprecipitation. The enriched mRNAs were subsequently purified and subjected to RNA sequencing, enabling transcriptomic profiling specifically within odor-activated neuronal populations, particularly olfactory sensory neurons. This approach provides a targeted view of gene expression programs induced by diverse odor stimulation paradigms in the heterogeneous olfactory epithelium. Overall design: C57BL/6J wild-type male mice (8 weeks old) were individually housed in sealed, odor-proof containers and habituated in an odorless environment for 4 hours prior to stimulation. Mice were then exposed for 90 minutes to one of the following conditions: single odorants (acetophenone, decanal, octanal, cis-3-hexenol), binary mixtures (acetophenone–decanal, octanal–cis-3-hexenol), or complex fragrances (floral, mint, floral–mint). Immediately following exposure, olfactory epithelium tissues were collected, perfused, and homogenized. pS6-associated ribosome complexes were isolated from the homogenates through immunoprecipitation, selectively enriching ribosome-bound mRNAs from odor-activated cells. Three biological replicates were obtained for each condition, and the immunoprecipitated mRNAs were subsequently purified and processed for RNA sequencing.