GSM8726171: RiboTag, CA1, Opto, Input, replicate 3; Mus musculus; RNA-Seq
30 minutes after optogenetic activation, tissue was dissected from acute brain slices in HBSS with 0.1 mg/mL cycloheximide and then resuspended in 5% w/v ice-cold polysome buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM DTT) freshly supplemented with 40 U/ml RNasin Plus (Promega), cOmplete protease inhibitors (Roche), 0.1 mg/mL cycloheximide, and 0.5 mM DTT) and homogenized by dounce homogenization (40 strokes type A pestle followed by 40 strokes type B pestle). NP-40 was added to 1% final concentration and lysate was incubated on ice for 10 min. Supernatant was subsequently centrifuged at 2000 x g for 10 min at 4°C followed by 20,000 x g for 10 min at 4°C. 10% of the resulting lysate was used as input and the remaining lysate was pre-cleared by rotation with 50 μl Protein G Dynabeads (Invitrogen) (washed in polysome buffer) for 45 min at 4°C. HA-tagged ribosomes were collected by indirect IP by adding mouse anti-HA (8 μl antibody per mL lysate; Biolegend; MMS-101R) antibody directly to the lysate, rotating the mixture at 4°C for 4 hours, and adding 150 μl Protein G dynabeads to the lysate + antibody followed by overnight rotation at 4°C. Beads were washed twice with polysome buffer containing 1% NP-40 and three times with high salt wash buffer (20 mM HEPES, 5 mM MgCl2, 350 mM KCl, 1% NP-40) freshly supplemented with 0.1 mg/mL cycloheximide, and 1 mM DTT. All washes were 5 minutes with rotation at 4°C. RNA was extracted from input and IP samples by incubation with 1 mL Trizol at room temperature for 5 minutes and then RNeasy Lipid Tissue Mini Kit (Qiagen) via the manufacturer's protocol with on-column DNase treatment (Roche). RNA quantity and quality were obtained by the Agilent 2100 Pico Bioanalyzer system. The libraries were prepared by Illumina Stranded Total RNA Prep with Ribo-Zero Plus following the manufacturer's instructions. High-throughput sequencing was performed on NovaSeq (Illumina) to obtain 150 nucleotide paired-end reads.