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Updated: Mar 15, 2026

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

Published on: August 7, 2021

Ribonuclease selection for ribosome profiling.

Maxim V Gerashchenko¹, Vadim N Gladyshev²

¹Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Nucleic Acids Research

|September 18, 2016

Summary

This summary is machine-generated.

Choosing the right ribonuclease is crucial for accurate ribosome profiling. Ribonuclease T1 is recommended for preserving ribosomal integrity and converting polysomes to monosomes across diverse species.

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Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Ribosome profiling is vital for studying gene translation globally.
Methodological inconsistencies and analytical challenges hinder reproducibility in ribosome profiling.
Nuclease treatment is a critical step for ribosome-mRNA complex stability and polysome-to-monosome conversion.

Purpose of the Study:

To compare the efficacy of different ribonucleases (I, A, S7, T1) in ribosome profiling.
To evaluate nuclease performance across various model organisms (yeast, mice, bacteria, nematodes, fruit flies).
To identify the optimal ribonuclease for consistent and accurate ribosome profiling.

Main Methods:

Comparative ribosome profiling experiments were conducted.
Various ribonucleases were tested on ribosome-mRNA complexes.
Nuclease cutting preferences, periodicity patterns, and coverage were analyzed.
Polysome-to-monosome conversion efficiency was assessed across multiple species.

Main Results:

Different ribonucleases showed varying cutting preferences and coverage patterns.
Most tested ribonucleases yielded comparable gene expression estimates when ribosomal integrity was maintained.
Ribonuclease T1 was the only enzyme that consistently preserved ribosomal integrity and effectively converted polysomes to monosomes in all tested species.
Some ribonucleases led to complete degradation of ribosome populations.

Conclusions:

Ribonuclease selection significantly impacts ribosome profiling outcomes.
Ribonuclease T1 is the most reliable enzyme for preserving ribosomal integrity and achieving efficient polysome-to-monosome conversion across diverse model systems.
This study provides essential guidance for selecting appropriate ribonucleases in ribosome profiling experiments.