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A rapid protocol for ribosome profiling of low input samples

Andreas Meindl, Markus Romberger, Gerhard Lehmann, Norbert Eichner, Leon Kleemann, Jie Wu, Johannes Danner, Maria Boesl, Mikhail Mesitov, Gunter Meister, Julian König, Sebastian Leidel, View ORCID ProfileJan Medenbach
doi: https://doi.org/10.1101/2022.09.23.509038
Andreas Meindl
1Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany
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Markus Romberger
1Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany
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Gerhard Lehmann
2Biochemistry I, University of Regensburg, Regensburg, Germany
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Norbert Eichner
2Biochemistry I, University of Regensburg, Regensburg, Germany
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Leon Kleemann
3Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland
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Jie Wu
3Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland
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Johannes Danner
2Biochemistry I, University of Regensburg, Regensburg, Germany
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Maria Boesl
1Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany
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Mikhail Mesitov
4Institute of Molecular Biology (IMB), Mainz, Germany
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Gunter Meister
2Biochemistry I, University of Regensburg, Regensburg, Germany
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Julian König
4Institute of Molecular Biology (IMB), Mainz, Germany
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Sebastian Leidel
3Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland
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Jan Medenbach
1Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany
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  • ORCID record for Jan Medenbach
  • For correspondence: Jan.Medenbach@ur.de
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Abstract

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected from nucleolytic digestion by ribosomes. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of RNA. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

Competing Interest Statement

JM is a consultant to siTOOLs Biotech GmbH, Martinsried, Germany

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted September 23, 2022.
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A rapid protocol for ribosome profiling of low input samples
Andreas Meindl, Markus Romberger, Gerhard Lehmann, Norbert Eichner, Leon Kleemann, Jie Wu, Johannes Danner, Maria Boesl, Mikhail Mesitov, Gunter Meister, Julian König, Sebastian Leidel, Jan Medenbach
bioRxiv 2022.09.23.509038; doi: https://doi.org/10.1101/2022.09.23.509038
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A rapid protocol for ribosome profiling of low input samples
Andreas Meindl, Markus Romberger, Gerhard Lehmann, Norbert Eichner, Leon Kleemann, Jie Wu, Johannes Danner, Maria Boesl, Mikhail Mesitov, Gunter Meister, Julian König, Sebastian Leidel, Jan Medenbach
bioRxiv 2022.09.23.509038; doi: https://doi.org/10.1101/2022.09.23.509038

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