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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

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

Published: August 7th, 2021

DOI:

10.3791/62544

1Laboratory of Gene Expression, ECOTECH-Complex, Maria Curie-Sklodowska University, 2Department of Molecular Biology, Institute of Biological Sciences, Maria Curie-Sklodowska University, 3Faculty of Information Technology, Polish-Japanese Academy of Information Technology

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.

In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.

Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer's guidelines with some degree of optimization.

The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

The ribosome profiling technique (RIBO-seq) was developed in the laboratory of Jonathan Weissman at the University of California, San Francisco1. In comparison to other methods used to study gene expression at the translational level, RIBO-seq focuses on each ribosome binding to mRNA and provides information about its location and the relative number of ribosomes on a transcript. It enables monitoring the process of protein synthesis in vivo and can provide single codon resolution and accuracy allowing the measurement of the ribosome density on both, the individual mRNA and along the entire transcriptome in the cell. At the foundation ....

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1. Sample collection

  1. Prepare a bacterial culture. We recommend a culture volume of 100 mL per sample.
  2. Prepare equipment and reagents for sample collection: two scoopulas per sample, sterile 0.45 µm mixed cellulose esters membrane (MCE) filters, 50 mL sterile tubes, liquid nitrogen, 50 mg/mL chloramphenicol in 70% (vol/vol) ethanol (optional). Puncture the lid of 50 mL tubes to allow liquid nitrogen evaporation and prevent the explosion of closed tubes. Decontaminate scoopulas with laboratory de.......

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The exemplary results presented here were obtained in a study examining translation regulation in sporulating WT Bacillus subtilis cells. Overnight cultures were diluted to OD600 equal to 0.1 in 100 mL of rich medium and incubated at 37 °C with vigorous shaking until OD600 reached 0.5-0.6. The rich medium was then replaced with minimal medium to induce sporulation process and the incubation was continued for up to four hours. Cells were harvested every hour beginning with T0 - sporulat.......

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The key technical challenge of the ribosome profiling is the need to rapidly inhibit translation in order to capture a snapshot of ribosomes on mRNAs at a particular physiological state of interest. To accomplish this, translation inhibitors, rapid harvesting and flash freezing in liquid nitrogen are commonly used. Applying antibiotics is optional since they can cause artifacts. Chloramphenicol is a commonly used drug to arrest elongating ribosomes in bacterial RIBO-seq. However, it does not prevent initiation, resulting.......

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ALS would like to acknowledge the financial support of EMBO Installation Grants IG 3914, and POIR. 04.04.00-00-3E9C/17-00 carried out within the First TEAM programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

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Name Company Catalog Number Comments
10X TBE (powder) Invitrogen AM9864
2-Mercaptoethanol, 99%, pure Acros Organics 125472500
Adenosine 5'-Triphosphate (ATP) New England Biolabs P0756S
Aluminium oxide calcinated pure p.a. Chempur 114560600
Calcium chloride dihydrate Sigma-Aldrich C3881-500G
Chloramphenicol MP Biomedicals 190321
DNA Clean & Concentrator -5 Zymo Research D4004
Dnase I recombinant, Rnase-free Roche 4716728001
EDTA disodium salt Fisher Scientific E/P140/48
Ethyl Alcohol Absolut 99,8%  Pure-P.A.-Basic POCH Avantor Performance Materials Poland S.A BA6480111
Filtration apparatus VWR Collection 511-0265 all-glass filtration apparatus, with funnel, fritted base, cap, 47 mm Ø spring clamp and ground joint flask
Gel 40 (19:1) Rotiphorese 3030.1
Gel Loading Dye, Blue, 6X New England Biolabs E6138G
Guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate Sigma-Aldrich G0635-25MG
labZAP A&A Biotechnology 040-500
Magnesium acetate tetrahydrate Sigma-Aldrich M5661-250G
MCE membrane fiter Alfatec Technology M47MCE45GWS pore size: 0.45um
MICROBExpress Bacterial mRNA Purification Invitrogen AM1905
Multiplex Small RNA Library Prep Set for Illumina New England Biolabs E7300S
Nuclease-Free Water Ambion AM9937
Potassium Acetate Anhydrous Pure P.A. POCH Avantor Performance Materials Poland S.A 744330113
Quick-Load pBR322 DNA-MspI Digest New England Biolabs E7323A
RNA Clean & Concentrator -25 Zymo Research R1018
Sodium acetate Sigma-Aldrich S2889-250G
Sodium carbonate Sigma-Aldrich 223530-500G
Sodium hydrogen carbonate pure p.a. POCH Avantor Performance Materials Poland S.A 810530115
SYBR Gold nucleic acid gel stain Life Technologies S11494
T4 Polynucleotide Kinase New England Biolabs M0201L
T4 Polynucleotide Kinase Reaction Buffer New England Biolabs B0201S
TBE-Urea Sample Buffer (2x) Invitrogen LC6876
Tris(hydroxymethyl)amino-methane, ultrapure, 99,9% AlfaAesar J65594
Triton X-100, 98% Acros Organics 327371000
Urea G.R. lach:ner 40096-AP0

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