Methods Protoc. 2026 Mar 10. pii: 45. [Epub ahead of print]9(2):
Ribosome profiling, or Ribo-Seq, is a powerful tool for studying translation. It maps the positions of translating ribosomes on mRNAs, providing insights into actively expressed genes. Unlike mass spectrometry, Ribo-Seq is not affected by the same biases that limit mass spectrometry, such as protein size, concentration, trypsin digestibility, or hydrophobicity. Thus, the translatome has previously been used to discover unannotated genes, including small and overlapping ones that were missed by mass spectrometry or gene prediction models. However, a major limitation of classical ribosome profiling is its complexity, involving multiple steps such as sucrose density gradient centrifugation and gel electrophoresis. These make the method costly, time-consuming, and limit its throughput. Here, we compared the classical method using gradient centrifugation and size exclusion by gel electrophoresis with shortened versions to evaluate experimental performance and achieved reductions. Our results show that the sucrose density gradient centrifugation is essential for obtaining accurate Ribo-Seq data, whereas gel electrophoresis for size selection can be omitted (although this requires increased sequencing depth). Thus, future experiments can be conducted with reduced sample input and hands-on time while still achieving a reliable quantification of translation.
Keywords: Ribo-Seq; bacterial gene expression; microbial translatomics; ribosome profiling; shortened workflow; translation quantification