Conversion of Ribosome-Protected mRNA to DNA and Deep Sequencing for Ribosome Profiling in Haloferax volcanii
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature..
The translation of messenger RNA (mRNA) into protein is an essential process for all forms of life. The ability to monitor this process in a quantitative way by ribosome profiling-based approaches has revolutionized our ability to monitor protein synthesis in vivo and to explore and model complex cellular processes. Ribosome profiling is a high-throughput technique that globally analyzes the full set of ribosomes engaged in translation, providing insights into important aspects of the mechanism of protein synthesis and its regulation. This protocol covers the construction of a ribosome profiling library from culture harvesting, footprint isolation via ultracentrifugation, gel-based size fractionation, and footprint sequencing for a model halophilic archaeon, Haloferax volcanii. This approach has revealed the first global view of translation in the archaea.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2522 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2522(2022) vom: 20., Seite 223-242 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Gelsinger, Diego Rivera [VerfasserIn] |
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| Links: |
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| Themen: |
9007-49-2 |
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| Anmerkungen: |
Date Completed 23.09.2022 Date Revised 26.09.2022 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-2445-6_14 |
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| funding: |
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| 520 | |a The translation of messenger RNA (mRNA) into protein is an essential process for all forms of life. The ability to monitor this process in a quantitative way by ribosome profiling-based approaches has revolutionized our ability to monitor protein synthesis in vivo and to explore and model complex cellular processes. Ribosome profiling is a high-throughput technique that globally analyzes the full set of ribosomes engaged in translation, providing insights into important aspects of the mechanism of protein synthesis and its regulation. This protocol covers the construction of a ribosome profiling library from culture harvesting, footprint isolation via ultracentrifugation, gel-based size fractionation, and footprint sequencing for a model halophilic archaeon, Haloferax volcanii. This approach has revealed the first global view of translation in the archaea | ||
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