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Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

View ORCID ProfileErika C Urdaneta, Carlos H Vieira-Vieira, View ORCID ProfileTimon Hick, Hans-Herrmann Wessels, Davide Figini, Rebecca Moschall, View ORCID ProfileJan Medenbach, Uwe Ohler, Sander Granneman, Matthias Selbach, View ORCID ProfileBenedikt M Beckmann
doi: https://doi.org/10.1101/333385
Erika C Urdaneta
1IRI Life Sciences, Humboldt University, 10115 Berlin, Germany
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Carlos H Vieira-Vieira
2Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany
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Timon Hick
1IRI Life Sciences, Humboldt University, 10115 Berlin, Germany
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Hans-Herrmann Wessels
3Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
4Department of Biology, Humboldt University, 10115 Berlin, Germany
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Davide Figini
1IRI Life Sciences, Humboldt University, 10115 Berlin, Germany
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Rebecca Moschall
5Biochemistry I, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
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Jan Medenbach
5Biochemistry I, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
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Uwe Ohler
3Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany
4Department of Biology, Humboldt University, 10115 Berlin, Germany
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Sander Granneman
6Centre for Systems and Synthetic Biology (SynthSys), University of Edinburgh, Max Born Crescent, EH9 3BF, Edinburgh, UK
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Matthias Selbach
2Max-Delbrück-Center for Molecular Medicine, 13125 Berlin, Germany
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Benedikt M Beckmann
1IRI Life Sciences, Humboldt University, 10115 Berlin, Germany
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  • ORCID record for Benedikt M Beckmann
  • For correspondence: benedikt.beckmann@iri-lifesciences.de
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Abstract

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

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Posted May 30, 2018.
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Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction
Erika C Urdaneta, Carlos H Vieira-Vieira, Timon Hick, Hans-Herrmann Wessels, Davide Figini, Rebecca Moschall, Jan Medenbach, Uwe Ohler, Sander Granneman, Matthias Selbach, Benedikt M Beckmann
bioRxiv 333385; doi: https://doi.org/10.1101/333385
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Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction
Erika C Urdaneta, Carlos H Vieira-Vieira, Timon Hick, Hans-Herrmann Wessels, Davide Figini, Rebecca Moschall, Jan Medenbach, Uwe Ohler, Sander Granneman, Matthias Selbach, Benedikt M Beckmann
bioRxiv 333385; doi: https://doi.org/10.1101/333385

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