Am8670

Total RNA isolated from the sucrose gradient fractions was resolved using a 10% polyacrylamide gel in 0.5x TBE buffer buffer (1x TBE buffer contains 89 mM Tris, 89 mM boric acid, and 2 mM EDTA) and electroblotted onto a nylon (N+) membrane (GE Healthcare RPN203B) at 20 V for 90 min at 4°C in 0.5x TBE buffer. The membrane was crosslinked and pre-hybridized in UltraHyb Hybridization Solution (Thermo AM8670) at 42°C for 1 hour, then incubated overnight with 50 pmol Met-tRNA_i specific probe (5′-TGGTAGCAGAGGATGGTTTCGAT-3′). The probe was labeled on the 5′ end with [γ- 32P] ATP (Perkin Elmer) using T4 polynucleotide kinase (NEB M0201) according to the manufacturer’s protocol. Membranes were washed twice by 20 mL 6x SSC for 5 min at 42°C and twice by 20 mL 2x SSC and twice by 20 mL 1x SSC (20x SSC contains 0.3 M sodium citrate in 3 M NaCl). Membranes were then wrapped in saran wrap, exposed to a phosphor screen overnight, and visualized by phoshor-imaging.

For the preparation of RNA probes, 5 µg of template DNA amplified with DIG-tag primers (Supplementary table I) were transcribed with T7 polymerase from the DIG RNA labeling kit (11175025910, Roche, CH) for 2 h at 37 °C. Residual DNA was digested with DNase I for 15 min at 37 °C. DIG-labeled RNA probes were isolated using phenol–chloroform extraction and eluted in 25 µL EB (19086, Qiagen, DE). For northern blotting, 8 µg of RNA were loaded on a 1.2% agarose gel in 1X MOPS buffer (AM8671, Thermo Scientific, US) and RNA was separated by size through electrophoresis at 100 V. After electrophoresis, agarose gel was blotted onto positively charged nylon membrane (11209299001, Roche, CH). The RNA was then UV cross-linked to the membrane and pre-hybridized for 30 min at 68 °C with ultrasensitive hybridization buffer (AM8670, Thermo Scientific, US). The membrane was incubated with RNA probes at 50 ng/mL hybridization buffer over night at 68 °C. The membrane was then washed with high and low stringency wash buffer prior to blocking in 1X blocking solution from DIG wash and block buffer set (11585762001, Roche, CH). After blocking, membrane was incubated with 1:5000 Anti-DIG AP from DIG Luminescent Detection Kit (11363514910, Roche, CH). CSPD was used as a substrate for chemiluminescent detection.