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. 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 binary odor mixtures delivered by two different methods. In the “+” condition, both odorants were applied to a single piece of filter paper, allowing direct mixing prior to volatilization. In the “&” condition, the odorants were applied separately to two filter papers placed in close proximity (~1 mm apart), enabling mixing in the air without physical contact on the filter. These procedures generated four binary mixture groups (A+D, A&D, O+C, and O&C), along with a no-odor control. Immediately following stimulation, 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.