GSM8969047: C57BL/6J, Olfactory epithelium, pS6-IP, AD condition, control 2; Mus musculus; RNA-Seq
Olfactory epithelium tissues were rapidly collected from mice immediately after odorant exposure and perfused with chilled buffer (PBS, 5mM NaF, 2.5mM Na3VO4, 2.5mM Na4P2O7, 5mM β-glycerophosphate, 100μg/mL cycloheximide, protease inhibitor). The tissues were homogenized in a buffer containing 10mM HEPES (pH 7.4), 150mM KCl, 10mM MgCl2, 100mM calyculin A, 0.5mM DTT, 100U/mL RNasin, 250μg/mL cycloheximide, and protease inhibitors using a TissueLyser II (Qiagen) for 2 minutes at 20Hz. Homogenates were centrifuged at 2,000×g for 10 minutes at 4°C. The supernatant was supplemented with 0.7% NP-40, incubated on ice, and centrifuged at 20,000×g for 10 minutes at 4°C. For pS6-immunoprecipitation, the supernatant was incubated with Protein A Dynabeads pre-coupled to anti-phospho-S6 antibody (Invitrogen 44-923G) for 1 hour at room temperature. After four washes with high-salt buffer (10 mM HEPES (pH 7.4), 350 mM KCl, 5 mM MgCl2, 2 mM DTT, 1% NP-40, 100 U/mL RNasin, and 100 μg/mL cycloheximide), RNA was eluted with Buffer RLT and purified using the RNeasy Mini Kit (Qiagen) with on-column DNase digestion. Libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit (Illumina), and sequencing was carried out on an Illumina NovaSeq 6000 platform according to the manufacturer's protocols.