title: GSM9244070: Ribo NCD 5; Mus musculus; RNA-Seq
library_construction_protocol: The olfactory epithelium was dissected on ice in buffer containing 1×HBSS (Gibco, with Ca2+ and Mg2+), 2.5 mM HEPES (pH 7.4), 35 mM glucose, 100 μg/ml cycloheximide, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM beta-glycerophosphate. 4 epithelia were pooled for homogenization at 250 rpm for 10 times, and 9 times at 750 rpm on a rotating glass/teflon potter homogenizer (Potter S, Braun) at 4°C in 1 ml of buffer containing 150 mM KCl, 5 mM MgCl2, 10 mM HEPES, 100 nM Calyculin A, 2 mM DTT, 100 U RNasin (Promega), 100 μg cycloheximide, 1 tablet of Complete mini EDTA-free protease inhibitor cocktail/7 ml and 1 tablet PhosSTOP/5 ml. Samples were spun in 1.5-ml LoBind tubes (Eppendorf), and centrifuged at 2,000g for 10 min at 4°C. 90 μl 10% NP-40 (v/v) and 90 μl 300 mM DHPC (Avanti Polar Lipids) were added to the supernatant which was centrifuged again at 17,000g for 10 min at 4°C. The supernatant was transferred to a new LoBind tube, and mixed with 20 μl pS6 antibody (#2215, Cell Signaling). Protein A Dynabeads (Invitrogen, 100 μl per sample) were washed 3 times with 900 μl buffer containing 150 mM KCl, 5 mM MgCl2, 10 mM HEPES, 0.05% BSA (w/v) and 1% NP-40. After antibody binding, the mixture was added to the washed beads mixed by pipetting, followed by incubation for 1 h at 4°C on a rotator. After incubation, the ribosome-bound beads were washed 4 times with 700 μl buffer containing 350 mM KCl, 5 mM MgCl2, 10 mM HEPES, 1% NP-40, 2 mM DTT, 100 U/ml recombinant RNasin (Promega), 100 μg/ml cycloheximide, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM beta-glycerophosphate. During the final wash, beads were placed onto the magnet and moved to room temperature. After removing supernatant, RNA was eluted by mixing the beads with 350 μl RLT (from Qiagen RNeasy kit). The eluted RNA was purified using RNeasy Micro kit (Qiagen). For library preparation, the Illumina Nextera XT DNA sample preparation protocol was used, with 1 ng cDNA input
identifiers.namespace: GEO
identifiers.id: GSM9244070_r1