pS6-IP RNA-seq of mouse olfactory epithelium upon binary odor mixture stimulation under different delivery methods

Phosphorylation of ribosomal protein S6 (pS6) serves as a molecular marker of neuronal activation by external stimuli. In this study, olfactory epithelium tissues were obtained from mice exposed to binary odor mixtures consisting of acetophenone–decanal or octanal–cis-3-hexenol. To examine how different delivery methods of odor presentation influence neural responses, two paradigms for binary mixtures were employed. In the “+” condition, the two odorants were directly combined on a single piece of filter paper, allowing interaction prior to volatilization. In contrast, in the “&” condition, the odorants were applied separately to two filter papers placed in close proximity (~1 mm apart), such that the components mixed only in the air upon evaporation. Using these approaches, four experimental odor groups were generated (A+D, A&D, O+C, and O&C), along with a no-odor control, resulting in a total of five groups. Following stimulation, pS6-associated ribosome complexes were isolated from olfactory epithelium lysates through immunoprecipitation, selectively enriching mRNAs undergoing active translation in odor-activated cells. RNA sequencing of these samples enabled transcriptomic profiling within activated neuronal populations, providing insights into gene expression programs associated with distinct delivery methods of binary odor stimulation.