Epitranscriptomic mapping of RNA modifications at single-nucleotide resolution using rhodamine sequencing (Rho-seq)

Olivier Finet; Carlo Yague-Sanz; Damien Hermand

doi:10.1016/j.xpro.2022.101369

Epitranscriptomic mapping of RNA modifications at single-nucleotide resolution using rhodamine sequencing (Rho-seq)

University of Namur

Research output: Contribution to journal › Article › peer-review

Abstract

The recent development of epitranscriptomics revealed a new fundamental layer of gene expression, but the mapping of most RNA modifications remains technically challenging. Here, we describe our protocol for Rho-Seq, which enables the mapping of dihydrouridine RNA modification at single-nucleotide resolution. Rho-Seq relies on specific rhodamine-labeling of a subset of modified nucleotides that hinders reverse transcription. Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022).

Original language	English
Article number	101369
Journal	STAR Protocols
Volume	3
Issue number	2
DOIs	https://doi.org/10.1016/j.xpro.2022.101369
Publication status	Published - 17 Jun 2022

Funding

The photographs of RNA samples at various steps of the rhodamine labeling protocol (Figure 4) are courtesy of Florian Marchand. We also thank Valérie Migeot and Florian Marchand for critical reading of the protocol and helpful discussions. This work was facilitated by PomBase (www.pombase.org), the model organism database for the fission yeast Schizosaccharomyces pombe. O.F. and C.Y-S. were supported by FRIA fellowships. This work was supported by FNRS grants PDR T.0012.14, CDR J.0066.16, and PDR T.0112.2 to D.H. C.Y-S. is an FNRS Postdoctoral Researcher. D.H. is an FNRS Director of Research. O.F. developed and optimized the experimental part of the protocol. C.Y.-S. developed the computational part of the protocol and wrote the manuscript with inputs from all authors. D.H. conceptualized and supervised the study. All authors reviewed the manuscript. The authors declare no competing interests. The photographs of RNA samples at various steps of the rhodamine labeling protocol ( Figure 4 ) are courtesy of Florian Marchand. We also thank Valérie Migeot and Florian Marchand for critical reading of the protocol and helpful discussions. This work was facilitated by PomBase (www.pombase.org), the model organism database for the fission yeast Schizosaccharomyces pombe. O.F. and C.Y-S. were supported by FRIA fellowships. This work was supported by FNRS grants PDR T.0012.14 , CDR J.0066.16 , and PDR T.0112.2 to D.H. C.Y-S. is an FNRS Postdoctoral Researcher. D.H. is an FNRS Director of Research.

Keywords

Gene Expression
Molecular Biology
RNAseq
Sequence analysis

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10.1016/j.xpro.2022.101369

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title = "Epitranscriptomic mapping of RNA modifications at single-nucleotide resolution using rhodamine sequencing (Rho-seq)",

abstract = "The recent development of epitranscriptomics revealed a new fundamental layer of gene expression, but the mapping of most RNA modifications remains technically challenging. Here, we describe our protocol for Rho-Seq, which enables the mapping of dihydrouridine RNA modification at single-nucleotide resolution. Rho-Seq relies on specific rhodamine-labeling of a subset of modified nucleotides that hinders reverse transcription. Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022).",

keywords = "Gene Expression, Molecular Biology, RNAseq, Sequence analysis",

author = "Olivier Finet and Carlo Yague-Sanz and Damien Hermand",

note = "Funding Information: The photographs of RNA samples at various steps of the rhodamine labeling protocol (Figure 4) are courtesy of Florian Marchand. We also thank Val{\'e}rie Migeot and Florian Marchand for critical reading of the protocol and helpful discussions. This work was facilitated by PomBase (www.pombase.org), the model organism database for the fission yeast Schizosaccharomyces pombe. O.F. and C.Y-S. were supported by FRIA fellowships. This work was supported by FNRS grants PDR T.0012.14, CDR J.0066.16, and PDR T.0112.2 to D.H. C.Y-S. is an FNRS Postdoctoral Researcher. D.H. is an FNRS Director of Research. O.F. developed and optimized the experimental part of the protocol. C.Y.-S. developed the computational part of the protocol and wrote the manuscript with inputs from all authors. D.H. conceptualized and supervised the study. All authors reviewed the manuscript. The authors declare no competing interests. Funding Information: The photographs of RNA samples at various steps of the rhodamine labeling protocol ( Figure 4 ) are courtesy of Florian Marchand. We also thank Val{\'e}rie Migeot and Florian Marchand for critical reading of the protocol and helpful discussions. This work was facilitated by PomBase (www.pombase.org), the model organism database for the fission yeast Schizosaccharomyces pombe. O.F. and C.Y-S. were supported by FRIA fellowships. This work was supported by FNRS grants PDR T.0012.14 , CDR J.0066.16 , and PDR T.0112.2 to D.H. C.Y-S. is an FNRS Postdoctoral Researcher. D.H. is an FNRS Director of Research. Publisher Copyright: {\textcopyright} 2022 The Authors",

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AU - Finet, Olivier

AU - Yague-Sanz, Carlo

AU - Hermand, Damien

N1 - Funding Information: The photographs of RNA samples at various steps of the rhodamine labeling protocol (Figure 4) are courtesy of Florian Marchand. We also thank Valérie Migeot and Florian Marchand for critical reading of the protocol and helpful discussions. This work was facilitated by PomBase (www.pombase.org), the model organism database for the fission yeast Schizosaccharomyces pombe. O.F. and C.Y-S. were supported by FRIA fellowships. This work was supported by FNRS grants PDR T.0012.14, CDR J.0066.16, and PDR T.0112.2 to D.H. C.Y-S. is an FNRS Postdoctoral Researcher. D.H. is an FNRS Director of Research. O.F. developed and optimized the experimental part of the protocol. C.Y.-S. developed the computational part of the protocol and wrote the manuscript with inputs from all authors. D.H. conceptualized and supervised the study. All authors reviewed the manuscript. The authors declare no competing interests. Funding Information: The photographs of RNA samples at various steps of the rhodamine labeling protocol ( Figure 4 ) are courtesy of Florian Marchand. We also thank Valérie Migeot and Florian Marchand for critical reading of the protocol and helpful discussions. This work was facilitated by PomBase (www.pombase.org), the model organism database for the fission yeast Schizosaccharomyces pombe. O.F. and C.Y-S. were supported by FRIA fellowships. This work was supported by FNRS grants PDR T.0012.14 , CDR J.0066.16 , and PDR T.0112.2 to D.H. C.Y-S. is an FNRS Postdoctoral Researcher. D.H. is an FNRS Director of Research. Publisher Copyright: © 2022 The Authors

PY - 2022/6/17

Y1 - 2022/6/17

N2 - The recent development of epitranscriptomics revealed a new fundamental layer of gene expression, but the mapping of most RNA modifications remains technically challenging. Here, we describe our protocol for Rho-Seq, which enables the mapping of dihydrouridine RNA modification at single-nucleotide resolution. Rho-Seq relies on specific rhodamine-labeling of a subset of modified nucleotides that hinders reverse transcription. Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022).

AB - The recent development of epitranscriptomics revealed a new fundamental layer of gene expression, but the mapping of most RNA modifications remains technically challenging. Here, we describe our protocol for Rho-Seq, which enables the mapping of dihydrouridine RNA modification at single-nucleotide resolution. Rho-Seq relies on specific rhodamine-labeling of a subset of modified nucleotides that hinders reverse transcription. Although Rho-Seq was initially applied to the detection of dihydrouridine, we show here that it is applicable to other modifications including 7-methylguanosine or 4-thiouridine. For complete details on the use and execution of this protocol, please refer to Finet et al. (2022).

KW - Gene Expression

KW - Molecular Biology

KW - RNAseq

KW - Sequence analysis

UR - https://www.scopus.com/pages/publications/85129661951

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DO - 10.1016/j.xpro.2022.101369

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ER -

Epitranscriptomic mapping of RNA modifications at single-nucleotide resolution using rhodamine sequencing (Rho-seq)

Abstract

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Keywords

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