POL Scientific / JBM / Volume 10 / Issue 1 / DOI: 10.14440/jbm.2023.403
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PROTOCOLS

An experimental workflow for identifying RNA m6A alterations in cellular senescence by methylated RNA immunoprecipitation sequencing

Yue Shi2,3,  Zeming Wu1,2,  Weiqi Zhang2,3,5,  Jing Qu4,5,6,  Weimin Ci2,3,5,  Guang-Hui Liu1,4,5
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1 State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
2 CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China
3 China National Center for Bioinformation, Beijing 100101, China
4 Beijing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China
5 University of Chinese Academy of Sciences, Beijing 100049, China
6 State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
JBM 2023 , 10(1), 1;
Published: 25 August 2023
© 2023 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

N6-methyladenosine (m6A), the most prevalent mRNA modification in eukaryotic cells, is known to play regulatory roles in a wide array of biological processes, including aging and cellular senescence. To investigate such roles, the m6A modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-m6A antibody, followed by high-throughput sequencing (meRIP-seq or m6A-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA m6A landscape in senescent human cells. We described, in detail, sample preparation, mRNA isolation, immunoprecipitation, library preparation, sequencing, bioinformatic analysis and validation. We also provided tips and considerations for the optimization and interpretation of the results. Our protocol serves as a methodological resource for investigating transcriptomic m6A alterations in cellular senescence as well as a valuable paradigm for the validation of genes of interest.

Keywords
m6A
cellular senescence
meRIP-seq
bioinformatic analysis
qPCR
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TAB, Published by POL Scientific
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