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

Yue Shi; Zeming Wu; Weiqi Zhang; Jing Qu; Weimin Ci; Guang-Hui Liu

doi:10.14440/jbm.2023.403

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

J Biol Methods. 2023 Aug 25:10:e99010004. doi: 10.14440/jbm.2023.403. eCollection 2023.

Authors

Yue Shi^{1

2}, Zeming Wu^{3

4}, Weiqi Zhang^{1

2

5}, Jing Qu^{4

5

6}, Weimin Ci^{1

2

5}, Guang-Hui Liu^{3

4

5}

Affiliations

¹ CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
² China National Center for Bioinformation, Beijing 100101, China.
³ State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, 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.

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: bioinformatic analysis; cellular senescence; m6A; meRIP-seq; qPCR.