Ribo‐seq was performed as previously described [33 (link)]. SCC15 cells were first incubated with harringtonine (Solarbio) at a concentration of 2 μg/mL for 2 min, and then 100 μg/mL cycloheximide (Sigma‐Aldrich) was used to block translation. To prepare ribosome footprints, 300 μL lysate was mixed with 7.5 μL RNase I and 5 μL DNase I and incubated at 25°C for 45 min later. Nuclease digestion was stopped by adding 10 μL RNase inhibitor (Vazyme). Then, 100 μL of digested ribosome footprints was added to the column and centrifuged at 600 ×g for 2 min. Ribo‐seq libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (New England Biolabs).
The Ribo‐seq data were analyzed using the RiboTool kit (https://bioinformatics.sc.cn/RiboToolkit ) [34 (link)]. The translation efficiency (TE) was calculated as the ratio between the numbers of ribosome fragments aligned to the sequence coding for amino acids in protein (CDS) region and mRNA abundance for individual genes. Codon frequency analysis was performed after eliminating the genes with mRNA abundances lower than 5, and TEs with a 1‐fold decrease were considered TE‐downregulated genes.
The Ribo‐seq data were analyzed using the RiboTool kit (