Ribosome Sequencing for Translational Profiling

Ribosome sequencing (Ribo-Seq) was performed as previously described (22 (link)). Briefly, SCC25 cells were incubated with harringtonine (Solarbio), and then cycloheximide (Sigma-Aldrich) was used to block translation. After incubation with RNase I and DNase I at 25°C for 45 minutes, nuclease digestion was stopped with RNase inhibitor (Vazyme). Then, digested ribosome footprints were added to the column and centrifuged at 600g for 2 minutes. Ribo-Seq libraries were constructed using NEBNext R Multiple Small RNA Library Prep Set for Illumina R (New England Biolabs). The Ribo-Seq data were analyzed using the RiboTool kit (https://bioinformatics.sc.cn/RiboToolkit). Translation efficiency (TE) ratios between the numbers of ribosome fragments were calculated consistent with the sequence coding for amino acids in protein (CDS) region and mRNA abundance for individual genes. TEs with a 1-fold decrease were considered TE-downregulated genes.