GSM9590107: Transcripts from Scn10a+ neurons from LUAD lung, rep2; Mus musculus; RNA-Seq
RiboTag immunoprecipitations were performed as described previously (Sanz E, et al. Proc Natl Acad Sci, 2009), with some modifications. Initially, lung lobes from Scn10a-cre RiboTag mice, either from LUAD or naïve control mice, were homogenised using a tissue homogeniser with homogenization buffer in a 5% (w/v) ratio, containing 1% NP-40, 100 mM KCl, 50 mM Tris-HCl pH 7.5, and 12 mM MgCl2. This buffer was further enriched with 1% sodium deoxycholate (DOC), 100 µg/ml cycloheximide, a protease inhibitor cocktail, 1 mg/ml heparin, RNase inhibitors (200 U/mL), and 1 mM dithiothreitol (DTT). The resulting homogenates underwent centrifugation, and the supernatant was then incubated on a rotator at 4°C for 4 hours with 6 µl of anti-HA antibody (Biolegend, anti-HA #901513, 1:200). Subsequently, Pierce Protein A/G Magnetic Beads (Thermo Fisher, #88803) were bound to the antibody-ribosome complex through overnight incubation at 4°C on a rotator. The beads were then subjected to three washes with a high salt buffer comprising 300 mM KCl, 1% NP-40, 50 mM Tris-HCl pH 7.4, 12 mM MgCl2, 100 µg/ml cycloheximide, and 500 µM DTT. RNA was released from the ribosomes using 350 µl of RLT buffer (from Qiagen RNeasy kit) with 1% 2-mercaptoethanol. Subsequent RNA purification was performed using the RNeasy Plus Micro kit (Qiagen, 74034) following the manufacturer's instructions, and the eluted RNA was collected in 30 µl of RNase-free water and stored at -80°C. The Genomics Science Technology Platform (STP) at the Francis Crick Institute prepared sequencing libraries from at least 14 ng of total RNA using the Watchmaker mRNA Library Prep Kit with Polaris Depletion (Watchmaker, 7BK0002), in accordance with the manufacturer's instructions. Paired-end sequencing (2 × 100 bp) was performed on an Illumina NovaSeq X platform, targeting a minimum depth of 30 million reads per sample.