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

DOI:
10.3791/62544-v

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12:05 min

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August 07, 2021

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, , , ,
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

Chapters

  • 00:00Introduciton
  • 00:54Cell Harvesting and Lysis
  • 04:13MNase Digestion and Size Selection of Samples for RIBO-seq
  • 06:17rRNA Depletion and Alkaline Fragmentation of Samples for RNA-seq
  • 07:07Library Preparation and Size Selection of cDNA Libraries
  • 08:49Results: Quality of cDNA Libraries and Reads
  • 11:31Conclusion

Summary

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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.

Tags

RIBO-seqRibosome ProfilingProtein SynthesisTranslationRNA SequencingBacterial CultureSample CollectionLibrary PreparationNGS Sequencing

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