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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Updated: Oct 23, 2025

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

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

Natalia Kopik¹, Olga Chrobak¹, Przemyslaw Latoch²

¹Laboratory of Gene Expression, ECOTECH-Complex, Maria Curie-Sklodowska University; Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Sklodowska University.

Journal of Visualized Experiments : Jove

|August 23, 2021

Summary

This study simplifies ribosome profiling (RIBO-seq) for bacteria, enabling precise monitoring of protein synthesis. The streamlined protocol uses mechanical cell disruption and gel electrophoresis, reducing time and equipment needs for researchers.

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Last Updated: Oct 23, 2025

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

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

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Published on: April 14, 2016

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Published on: August 29, 2014

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Ribosome profiling (RIBO-seq) is crucial for studying protein synthesis in vivo.
Accurate mapping of ribosome positions on mRNA is essential for understanding translation.
Existing methods can be time-consuming and equipment-intensive.

Purpose of the Study:

To present a simplified and efficient protocol for ribosome profiling (RIBO-seq) in bacteria.
To detail sample preparation, cell harvesting, and mRNA isolation for RIBO-seq.
To reduce the time and equipment requirements for bacterial RIBO-seq.

Main Methods:

Rapidly inhibiting bacterial translation and harvesting cells via filtration and flash-freezing.
Mechanically disrupting cells using a mortar and pestle with aluminum oxide.
Isolating mRNA footprints using polyacrylamide gel electrophoresis (PAGE) without ultracentrifugation.
Preparing cDNA libraries using a commercial small RNA kit for Illumina sequencing.

Main Results:

The protocol yields 2 to 10 million uniquely mapped reads per sample.
The simplified method provides sufficient data for comprehensive bioinformatic analysis.
The protocol is quick, relatively easy, and uses standard laboratory equipment.

Conclusions:

This streamlined RIBO-seq protocol is effective for bacterial protein synthesis studies.
The method simplifies experimental execution and lowers resource demands.
The protocol facilitates wider adoption of RIBO-seq in bacterial research.