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Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0
BACKGROUND: RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex str...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9661767/ https://www.ncbi.nlm.nih.gov/pubmed/36372875 http://dx.doi.org/10.1186/s12915-022-01448-3 |
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author | Zhao, Jieyu Chow, Eugene Yui-Ching Yeung, Pui Yan Zhang, Qiangfeng Cliff Chan, Ting-Fung Kwok, Chun Kit |
author_facet | Zhao, Jieyu Chow, Eugene Yui-Ching Yeung, Pui Yan Zhang, Qiangfeng Cliff Chan, Ting-Fung Kwok, Chun Kit |
author_sort | Zhao, Jieyu |
collection | PubMed |
description | BACKGROUND: RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue. RESULTS: We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance. CONCLUSIONS: rG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-022-01448-3. |
format | Online Article Text |
id | pubmed-9661767 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-96617672022-11-15 Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 Zhao, Jieyu Chow, Eugene Yui-Ching Yeung, Pui Yan Zhang, Qiangfeng Cliff Chan, Ting-Fung Kwok, Chun Kit BMC Biol Methodology Article BACKGROUND: RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue. RESULTS: We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance. CONCLUSIONS: rG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12915-022-01448-3. BioMed Central 2022-11-13 /pmc/articles/PMC9661767/ /pubmed/36372875 http://dx.doi.org/10.1186/s12915-022-01448-3 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Article Zhao, Jieyu Chow, Eugene Yui-Ching Yeung, Pui Yan Zhang, Qiangfeng Cliff Chan, Ting-Fung Kwok, Chun Kit Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 |
title | Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 |
title_full | Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 |
title_fullStr | Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 |
title_full_unstemmed | Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 |
title_short | Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0 |
title_sort | enhanced transcriptome-wide rna g-quadruplex sequencing for low rna input samples with rg4-seq 2.0 |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9661767/ https://www.ncbi.nlm.nih.gov/pubmed/36372875 http://dx.doi.org/10.1186/s12915-022-01448-3 |
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