GSM8387347: Cortical neurons, Bicuculline CHX; Mus musculus; OTHER
Neurons were washed twice briefly with ice-cold PBS supplemented with 100 μg/ml cycloheximide. 400 μl cold cell lysis buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 5 mM, MgCl2, 500U/ml RNasin-Plus (Promega), 1x protease inhibitor cocktail (200X, Millipore), 100 μg/ml cycloheximide) was added directly to the cells, which were then scraped off of the dishes and transferred to 2 ml microcentrifuge tube containing Lysing Matrix-D (MP Biomedicals). Cells were lysed by vortexing 6 times (20 seconds each) with 40 s intervals on ice. After centrifugation for 10 min at 13,000 g at 4 °C, the supernatant was transferred into a new microcentrifuge tube and RNA concentrations of the cell lysates were measured using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher, Q32855). 10U RNase I (Thermo Fisher, EN0601) was added into cell lysate for every 100 μg RNA, and digestion was carried out at 4°C overnight with gentle rotation. The next day, RNase I digestion was stopped by adding Superase-In RNase inhibitor (Thermo Fisher, AM2696) at 1 U/μl. Digested cell lysate was loaded on top of 0.8 ml sucrose cushion (1 M sucrose, 20 mM HEPES, pH 7.4, 100 mM KCl, 5 mM, MgCl2, 100 μg/ml cycloheximide) in a 1.5 ml ultracentrifuge tube (Beckman Coulter, 343778) and centrifuged for 1.5 h at 75,000 rpm at 4°C using a TLA-120.2 rotor (Beckman Coulter). After carefully removing the supernatant, the ribosome pellet was solubilized in TRIzol (ThermoFisher) for RNA extraction. The extracted RNA was separated in a 15% Urea-TBE gel, and RNA fragments of 25 - 35 nt in length were excised for subsequent library construction.