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

Sci Rep. 2025 Jan 7;15(1):1058. doi: 10.1038/s41598-025-85150-8.

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.

Keywords: Human fetal brain; Human fetal liver; MeRIP Seq; Mouse brain; Mouse liver; m6A antibody.

Publication types

  • Comparative Study

MeSH terms

  • Adenosine* / analogs & derivatives
  • Animals
  • Antibodies* / genetics
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Immunoprecipitation / methods
  • Liver / metabolism
  • Mice
  • RNA / genetics
  • Sequence Analysis, RNA* / methods

Substances

  • N-methyladenosine
  • Adenosine
  • Antibodies
  • RNA