To prepare lysate for ribosome profiling,  200 µl glass beads and 500 µl of freshly prepared lysis buffer (composed of 20 mM Tris pH 8.0, 140 mM KCl, 1.5 mM MgCl2, 1% Triton X-100, 0.1 mg/mL cycloheximide, 2 mM DTT, and an EDTA-free protease inhibitor tablet) were added. The cells were pulverized by vortexing using a Multimixer for 2 minutes, followed by a 5-minute incubation on ice. This process was repeated three times to maximize the yield of RNA. After adding an additional 200 µl of lysis buffer to compensate for volume loss, the lysate was then centrifuged at 3000 g for 5 minutes at 4°C. After quantifying RNA concentration, per 200 µg RNA was incubated with 2 µl 100 U/µl RNase I (Ambion) for 1 h at 22 °C with gentle agitation (700 rpm) and the reaction was inhibited by the addition of 15 µl SuperaseIn (Thermo Fisher).   To perform polysome profiling,  another 50 µg RNA from the lysate was kept on ice undigested for the usage. Linear sucrose gradient centrifugation and fractionation were then performed as described previously [PMID: 24893926], except we modified the 10X sucrose gradient buffer (consisting of 0.5 M Tris-Acetate pH 7.0, 0.5 M NH4Cl, and 0.12 M MgCl2) to make it suitable for yeast cells.   Total RNA was isolated by the standard phenol: chloroform method as described in Zhang & Pelechano, 2021 (PMID:35474692) . RNA footprints were isolated at 20-35 nucleotide size using 15% TBE-urea gel. In order to further ligate 3' adapter and 5'adapter, RNA samples were treated with PNK to obtain 5' phosphorated and 3' hydroxylated ends, followed by ribosomal RNA depletion by using the RiboPools Depletion Kit (siTOOLs Biotech). Small RNA library was prepared by Small RNA Sample Preparation Kit for NEXTFLEX® following manufacture instructions. Sequencing was performed with single end setting, read length 47 bp on Illumina Nextseq 2000 sequencer. OTHER: Ribo-seq