Ribosome immunoprecipitation (IP)

The tissue samples were extracted from RiboTag^EC mice, flash-frozen in liquid nitrogen and then stored at −80°C. The samples were then homogenized on ice in ice-cold homogenization buffer (50 mM Tris, pH7.4, 100 mM KCl, 12 mM MgCl₂, 1% NP-40, 1 mM DTT, 1:100 protease inhibitor (Sigma), 200 units/mL RNasin (Promega) 1 mg/mL heparin and 0.1 mg/mL cycloheximide (Sigma) in RNase free DDW) 10% w/v with a Dounce homogenizer (Sigma) until the suspension was homogeneous. To remove cell debris, 1 mL of the homogenate was transferred to an Eppendorf tube and was centrifuged at 10,000xg and 4°C for 15 min. Supernatants were subsequently transferred to a fresh Eppendorf tube on ice, then 100 μL was removed for ‘input’ analysis and 3 μL (=3 µg) of anti-HA antibody (ab9110, Abcam) or 3 μL (=1 µg) of mouse monoclonal IgG1 antibody (Sigma, Cat# M5284) or 6 μL anti-RPL22 (Invitrogen Cat# PA5-68320) was added to the supernatant, followed by 1 hr of incubation with slow rotation in a cold room at 4°C. Meanwhile, Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific), 100 μL per sample, were equilibrated to homogenization buffer by washing three times. At the end of 1 hr of incubation with antibody, beads were added to each sample, followed by incubation 1 hr in cold room at 4°C. After that, samples were washed three times with high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:200 protease inhibitor, 100 units/mL RNasin and 0.1 mg/mL cycloheximide in RNase free DDW), 5 min per wash in a cold room on a rotator. At the end of the washes, beads were magnetized, and excess buffer was removed, 350 µL Lysis Buffer was added to the beads and RNA was extracted with RNeasy plus Mini kit (Qiagen). RNA was eluted in 30 μL H₂O and taken for RNA-Sequencing.