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An experimental workflow for identifying RNA m6A alterations in cellular senescence by methylated RNA
Yue Shi1,2, Zeming Wu3,4, Weiqi Zhang1,2,5
1CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, 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.
Insights
This study presents a detailed protocol for profiling N6-methyladenosine (m6A) RNA modifications in senescent human cells using meRIP-seq. The method enables comprehensive analysis of m6A changes during cellular senescence.
Area of Science:
- Molecular Biology
- Epigenetics
- Aging Research
Background:
- N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes.
- m6A plays crucial roles in biological processes, including aging and cellular senescence.
- Understanding m6A dynamics is vital for elucidating senescence mechanisms.
Purpose of the Study:
- To provide a detailed protocol for m6A profiling in senescent human cells using meRIP-seq.
- To establish a methodological resource for studying transcriptomic m6A alterations in cellular senescence.
- To offer guidance for optimizing and interpreting meRIP-seq results.
Main Methods:
- The protocol details sample preparation, mRNA isolation, and m6A immunoprecipitation (meRIP).
- It covers library preparation, high-throughput sequencing (m6A-seq), and bioinformatic analysis.
- Validation strategies for identified m6A modifications are also included.
Main Results:
- The presented protocol enables comprehensive profiling of the RNA m6A landscape in senescent cells.
- It facilitates the identification of specific m6A modification patterns associated with senescence.
- The method serves as a valuable tool for validating genes of interest.
Conclusions:
- This meRIP-seq protocol is a robust resource for investigating m6A alterations in cellular senescence.
- It provides a framework for understanding the functional impact of m6A modifications on senescence.
- The protocol can be adapted for studying m6A in other biological contexts.
