GSM4678242: LS-NTS Input Rep1; Mus musculus; RIP-Seq
Affinity purification of EGFP-tagged polysomes was done 3 or 4 weeks after virus injections (Nectow et al., 2017). Briefly, mice were separated into 3 biological replicate groups of 4-6 mice per group and euthanized. Brains were removed, and the lateral septum was dissected on ice and pooled. Tissue was homogenized in buffer containing 10 mM HEPES-KOH (pH 7.4), 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 100 μg/mL cycloheximide, RNasin (Promega) and SUPERase-In™ (Life Technologies) RNase inhibitors, and Complete-EDTA-free protease inhibitors (Roche) and then cleared by two-step centrifugation to isolate polysome-containing cytoplasmic supernatant. Polysomes were immunoprecipitated using monoclonal anti-EGFP antibodies (clones 19C8 and 19F7; see Heiman et al., 200830) bound to biotinylated-Protein L (Pierce; Thermo Fisher Scientific)-coated streptavidin-conjugated magnetic beads (Life Technologies). A small amount of tissue RNA was saved before the immunoprecipitation (Input) and both input and immunoprecipitated RNA (IP) were then purified using RNAeasy Mini kit (QIAGEN). RNA quality was checked using an RNA PicoChip on a bioanalyzer. RIN values >7 were used. Experiments were performed in triplicates for each group. cDNA was amplified using SMARTer Ultralow Input RNA for Illumina Sequencing Kit and sequenced on an Illumina HiSeq2500 platform.
GEO Accession
GSM4678242