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Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples
Jiafeng Lu1, Wenjuan Xia1, Jincheng Li1
1State Key Laboratory of Reproductive Medicine, Suzhou Municipal Hospital, Suzhou Affiliated Hospital of Nanjing Medical University, Gusu School, Nanjing Medical University, Suzhou, 215002, China.
Abstract:
The most effective method for mapping N6-methyladenosine (m6A) is m6A RNA immunoprecipitation sequencing (MeRIP-seq). The quality of MeRIP-seq relies on various factors, with the anti-m6A antibody being a crucial determinant. However, comprehensive research on anti-m6A antibody selection and optimal concentrations for different tissues has been limited. In this study, we optimized the concentration of five different anti-m6A antibodies across various tissues. Our findings demonstrated that 5 µg of Millipore antibodies (ABE572 and MABE1006) performed well, starting from 15 µg total RNA from the liver, while 1.25 µg of Cell Signaling Technology antibodies (CST) (#56593) was suitable for low-input total RNA. In summary, we provide a significant guideline for anti-m6A antibody selection in MeRIP sequencing for different tissues, especially in the context of low-input RNA.
Insights
Optimizing anti-m6A antibody concentration is key for high-quality m6A RNA immunoprecipitation sequencing (MeRIP-seq). This study provides guidelines for antibody selection and concentration across tissues, especially for low-input RNA samples.
Area of Science:
- Epigenetics and RNA modifications
Background:
- N6-methyladenosine (m6A) is a crucial epitranscriptomic mark.
- m6A RNA immunoprecipitation sequencing (MeRIP-seq) is the gold standard for mapping m6A modifications.
- Optimal anti-m6A antibody selection and concentration are critical for MeRIP-seq quality but lack comprehensive data across tissues.
Purpose of the Study:
- To optimize the concentration of five different anti-m6A antibodies for MeRIP-seq.
- To evaluate antibody performance across various tissue types.
- To provide guidelines for antibody selection, particularly for low-input RNA samples.
Main Methods:
- Testing five distinct anti-m6A antibodies.
- Optimizing antibody concentrations using varying amounts of total RNA from different tissues.
- Evaluating antibody performance based on MeRIP-seq quality metrics.
Main Results:
- Millipore antibodies (ABE572, MABE1006) required 5 µg for optimal performance with 15 µg total RNA from liver.
- Cell Signaling Technology (CST) antibody (#56593) at 1.25 µg was effective for low-input total RNA samples.
- Antibody performance varied depending on the specific antibody and tissue type.
Conclusions:
- Established guidelines for selecting and optimizing anti-m6A antibody concentrations for MeRIP-seq.
- Demonstrated tissue-specific optimization requirements for anti-m6A antibodies.
- Provided crucial recommendations for successful MeRIP-seq experiments, especially with limited RNA input.
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