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<p>The most effective method for mapping N6-methyladenosine (m<sup>6</sup>A) is m<sup>6</sup>A RNA immunoprecipitation sequencing (MeRIP-seq). The quality of MeRIP-seq relies on various factors, with the anti-m<sup>6</sup>A antibody being a crucial determinant. However, comprehensive research on anti-m<sup>6</sup>A antibody selection and optimal concentrations for different tissues has been limited. In this study, we optimized the concentration of five different anti-m<sup>6</sup>A antibodies across various tissues. Our findings demonstrated that 5&#xa0;µg of Millipore antibodies (ABE572 and MABE1006) performed well, starting from 15&#xa0;µg total RNA from the liver, while 1.25&#xa0;µg of Cell Signaling Technology antibodies (CST) (#56593) was suitable for low-input total RNA. In summary, we provide a significant guideline for anti-m<sup>6</sup>A antibody selection in MeRIP sequencing for different tissues, especially in the context of low-input RNA.</p>

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Comparative analysis of improved m6A sequencing based on antibody optimization for low-input samples

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年1月7日

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  • Jiafeng Lu,
  • Wenjuan Xia,
  • Jincheng Li,
  • Liya Zhang,
  • Chunfeng Qian,
  • Hong Li,
  • Boxian Huang

摘要

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.