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A low-cost, low-input method establishment for m6A MeRIP-seq
Wenjuan Xia1, Ling Guo1, Huapeng Su1
1State Key Laboratory of Reproductive Medicine and Offspring Health, Suzhou Affiliated Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, 215002, China.
Abstract:
N6-methyladenosine (m6A) is a highly prevalent modification found in mammal mRNA molecules that plays a crucial role in the regulation of cellular function. m6A RNA immunoprecipitation sequencing (MeRIP-seq) has been frequently used in transcriptomics research to identify the location of m6A. MABE572 (Millipore) is the most widely utilized and efficient anti-m6A antibody for MeRIP-seq. However, due to the high dose and price of this antibody, which has also been taken off the market, we discovered that CST's anti-m6A antibody can be used instead of MABE572 to map the m6A transcriptome. In the present study, we performed different concentrations of the CST anti-m6A antibodies with the corresponding initiation RNA of HEK293T cells, 2.5 μg antibody with 1 μg total RNA, 1.25 μg antibody with 0.5 μg total RNA, and 1.25 μg antibody with 0.1 μg total RNA. By comparing the m6A peak calling, enriched motifs, alternative splicing events, and nuclear transcripts modified by m6A between the CST and Millipore libraries, it was found that the CST library presented similar data to Millipore, even at incredibly low doses. The volume and cost of antibodies are significantly reduced by this refined MeRIP-seq using CST antibody, making it convenient to map future large-scale sample m6A methylation.
Insights
A new study shows CST's anti-m6A antibody effectively maps N6-methyladenosine (m6A) RNA modifications using MeRIP-seq. This cost-effective alternative to the discontinued Millipore antibody provides similar results at lower doses, enabling large-scale m6A transcriptome analysis.
Area of Science:
- Molecular Biology
- Epigenetics
- Transcriptomics
Background:
- N6-methyladenosine (m6A) is a prevalent mRNA modification regulating cellular functions.
- m6A RNA immunoprecipitation sequencing (MeRIP-seq) is crucial for identifying m6A locations.
- The widely used Millipore MABE572 anti-m6A antibody is expensive and discontinued.
Purpose of the Study:
- To evaluate CST's anti-m6A antibody as a cost-effective and accessible alternative for MeRIP-seq.
- To compare the efficacy of CST antibody-based MeRIP-seq with the MABE572 antibody.
- To optimize MeRIP-seq protocols for large-scale m6A transcriptome mapping.
Main Methods:
- Performed MeRIP-seq using varying concentrations of CST anti-m6A antibody with HEK293T cell RNA.
- Compared m6A peak calling, motif enrichment, and alternative splicing events between CST and Millipore libraries.
- Analyzed nuclear transcripts modified by m6A using both antibody types.
Main Results:
- CST antibody-based MeRIP-seq yielded comparable data to the Millipore MABE572 antibody.
- Effective m6A mapping was achieved even with significantly reduced amounts of CST antibody.
- The CST antibody significantly lowers the cost and volume requirements for MeRIP-seq.
Conclusions:
- CST's anti-m6A antibody is a viable and economical replacement for MABE572 in MeRIP-seq.
- This refined MeRIP-seq protocol facilitates large-scale m6A transcriptome analysis.
- Reduced antibody usage and cost make m6A mapping more accessible for researchers.
