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Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation
The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture and ultimately seque...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499544/ https://www.ncbi.nlm.nih.gov/pubmed/28449108 http://dx.doi.org/10.1093/nar/gkx286 |
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author | Routh, Andrew Ji, Ping Jaworski, Elizabeth Xia, Zheng Li, Wei Wagner, Eric J. |
author_facet | Routh, Andrew Ji, Ping Jaworski, Elizabeth Xia, Zheng Li, Wei Wagner, Eric J. |
author_sort | Routh, Andrew |
collection | PubMed |
description | The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3΄ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called ‘ClickSeq’. Here, we adapt this method to direct the cDNA synthesis specifically toward the 3΄UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA. |
format | Online Article Text |
id | pubmed-5499544 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-54995442017-07-10 Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation Routh, Andrew Ji, Ping Jaworski, Elizabeth Xia, Zheng Li, Wei Wagner, Eric J. Nucleic Acids Res Methods Online The recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3΄ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called ‘ClickSeq’. Here, we adapt this method to direct the cDNA synthesis specifically toward the 3΄UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA. Oxford University Press 2017-07-07 2017-04-26 /pmc/articles/PMC5499544/ /pubmed/28449108 http://dx.doi.org/10.1093/nar/gkx286 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://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 Routh, Andrew Ji, Ping Jaworski, Elizabeth Xia, Zheng Li, Wei Wagner, Eric J. Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation |
title | Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation |
title_full | Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation |
title_fullStr | Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation |
title_full_unstemmed | Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation |
title_short | Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation |
title_sort | poly(a)-clickseq: click-chemistry for next-generation 3΄-end sequencing without rna enrichment or fragmentation |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499544/ https://www.ncbi.nlm.nih.gov/pubmed/28449108 http://dx.doi.org/10.1093/nar/gkx286 |
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