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Chemical capping improves template switching and enhances sequencing of small RNAs

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing t...

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Autores principales: Wulf, Madalee G, Maguire, Sean, Dai, Nan, Blondel, Alice, Posfai, Dora, Krishnan, Keerthana, Sun, Zhiyi, Guan, Shengxi, Corrêa, Ivan R
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8754658/
https://www.ncbi.nlm.nih.gov/pubmed/34581823
http://dx.doi.org/10.1093/nar/gkab861
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author Wulf, Madalee G
Maguire, Sean
Dai, Nan
Blondel, Alice
Posfai, Dora
Krishnan, Keerthana
Sun, Zhiyi
Guan, Shengxi
Corrêa, Ivan R
author_facet Wulf, Madalee G
Maguire, Sean
Dai, Nan
Blondel, Alice
Posfai, Dora
Krishnan, Keerthana
Sun, Zhiyi
Guan, Shengxi
Corrêa, Ivan R
author_sort Wulf, Madalee G
collection PubMed
description Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.
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spelling pubmed-87546582022-01-13 Chemical capping improves template switching and enhances sequencing of small RNAs Wulf, Madalee G Maguire, Sean Dai, Nan Blondel, Alice Posfai, Dora Krishnan, Keerthana Sun, Zhiyi Guan, Shengxi Corrêa, Ivan R Nucleic Acids Res Methods Online Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit. Oxford University Press 2021-09-28 /pmc/articles/PMC8754658/ /pubmed/34581823 http://dx.doi.org/10.1093/nar/gkab861 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Methods Online
Wulf, Madalee G
Maguire, Sean
Dai, Nan
Blondel, Alice
Posfai, Dora
Krishnan, Keerthana
Sun, Zhiyi
Guan, Shengxi
Corrêa, Ivan R
Chemical capping improves template switching and enhances sequencing of small RNAs
title Chemical capping improves template switching and enhances sequencing of small RNAs
title_full Chemical capping improves template switching and enhances sequencing of small RNAs
title_fullStr Chemical capping improves template switching and enhances sequencing of small RNAs
title_full_unstemmed Chemical capping improves template switching and enhances sequencing of small RNAs
title_short Chemical capping improves template switching and enhances sequencing of small RNAs
title_sort chemical capping improves template switching and enhances sequencing of small rnas
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8754658/
https://www.ncbi.nlm.nih.gov/pubmed/34581823
http://dx.doi.org/10.1093/nar/gkab861
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