2.4. Ribosome Profiling (Ribo-seq)

Ribosome profiling was done as described before [11,41,42]. Three biological replicates (each consisting of material from at least three plants) for each treatment were analyzed as follows: 400 mg of deep-frozen, ground leaf material was thawed on ice in 5 mL of extraction buffer (200 mM Tris/HCl pH 8.0, 200 mM KCl, 35 mM MgCl₂, 0.2 M sucrose, 1% Triton X-100, 2% polyoxyethylen-10-tridecyl-ether, 5 mM dithiothreitol, 100 µg/mL chloramphenicol, 50 µg/mL cycloheximide). The extract was centrifuged for 5 min at 13,200 g and 4 °C. 600 µL of the supernatant was removed for analysis by RNA-seq (Section 2.5), and the remaining supernatant was centrifuged for 10 min at 15,000 g and 4 °C. CaCl₂ was added to the resulting supernatant to a concentration of 5 mM, followed by 750 units of micrococcal nuclease (Thermo Fisher Scientific, Waltham, MA, USA), and the mixture was incubated for 1 h at room temperature. The digested extract was loaded on a 2-mL sucrose cushion (40 mM Tris/acetate pH 8.0, 100 mM KCl, 15 mM MgCl₂, 1 M sucrose, 5 mM dithiothreitol, 100 µg/mL chloramphenicol, 50 µg/mL cycloheximide) and centrifuged for 3 h at 55,000 g and 4 °C in a Type 70 Ti rotor (Beckman Coulter, Brea, CA, USA). The pellet was dissolved in 1% SDS, 10 mM Tris/HCl pH 8.0, and 1 mM EDTA. RNA was purified using the PureLink miRNA Isolation Kit (Thermo Fisher Scientific). The 16 to 42-nt fraction was isolated by electrophoresis and treated with T4 polynucleotide kinase before library preparation using the TruSeq Small RNA Library Preparation Kit (Illumina, San Diego, CA, USA). Sequencing was performed on the HiSeq 4000 platform (Illumina).