Abridged Ribosome Profiling for Accurate Bacterial Translation Measurements

TL;DR

This paper introduces a simplified ribosome profiling method for bacteria that reduces time and cost while maintaining accuracy.

Contribution

The study identifies that gel electrophoresis can be omitted in ribosome profiling if sequencing depth is increased, streamlining the workflow.

Findings

Sucrose density gradient centrifugation is essential for accurate Ribo-Seq data.

Gel electrophoresis can be omitted if sequencing depth is increased.

Simplified protocols reduce time and sample input while maintaining reliable translation quantification.

Abstract

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.

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