Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing

Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing, which excludes truncated RNAs from the measurement. We describe steps for preparing recombinant e...

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Detalles Bibliográficos
Autores principales: Ogami, Koichi, Oishi, Yuka, Hoshino, Shin-ichi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10239010/
https://www.ncbi.nlm.nih.gov/pubmed/37243600
http://dx.doi.org/10.1016/j.xpro.2023.102340
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author Ogami, Koichi
Oishi, Yuka
Hoshino, Shin-ichi
author_facet Ogami, Koichi
Oishi, Yuka
Hoshino, Shin-ichi
author_sort Ogami, Koichi
collection PubMed
description Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing, which excludes truncated RNAs from the measurement. We describe steps for preparing recombinant eIF4E mutant protein, purifying m7G- capped RNAs, library preparation, and sequencing. Resulting data can be used not only for expression profiling and poly(A) tail length estimation but also for detecting alternative splicing and polyadenylation events and RNA base modification. For complete details on the use and execution of this protocol, please refer to Ogami et al. (2022).(1)
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spelling pubmed-102390102023-06-04 Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing Ogami, Koichi Oishi, Yuka Hoshino, Shin-ichi STAR Protoc Protocol Poly(A) tail metabolism contributes to post-transcriptional regulation of gene expression. Here, we present a protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing, which excludes truncated RNAs from the measurement. We describe steps for preparing recombinant eIF4E mutant protein, purifying m7G- capped RNAs, library preparation, and sequencing. Resulting data can be used not only for expression profiling and poly(A) tail length estimation but also for detecting alternative splicing and polyadenylation events and RNA base modification. For complete details on the use and execution of this protocol, please refer to Ogami et al. (2022).(1) Elsevier 2023-05-26 /pmc/articles/PMC10239010/ /pubmed/37243600 http://dx.doi.org/10.1016/j.xpro.2023.102340 Text en © 2023 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Ogami, Koichi
Oishi, Yuka
Hoshino, Shin-ichi
Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing
title Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing
title_full Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing
title_fullStr Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing
title_full_unstemmed Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing
title_short Protocol for analyzing intact mRNA poly(A) tail length using nanopore direct RNA sequencing
title_sort protocol for analyzing intact mrna poly(a) tail length using nanopore direct rna sequencing
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10239010/
https://www.ncbi.nlm.nih.gov/pubmed/37243600
http://dx.doi.org/10.1016/j.xpro.2023.102340
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