2. Academic Validation
  3. Mild and ultrafast GLORI enables absolute quantification of m6A methylome from low-input samples

Mild and ultrafast GLORI enables absolute quantification of m6A methylome from low-input samples

  • Nat Methods. 2025 Jun;22(6):1226-1236. doi: 10.1038/s41592-025-02680-9.
Hanxiao Sun # 1,  Bo Lu # 1,  Zeyu Zhang # 2,  Ye Xiao 3 4,  Zhe Zhou 1,  Lin Xi 3,  Zhichao Li 1,  Zhe Jiang 1,  Jiayi Zhang 1,  Meng Wang 2,  Cong Liu 1,  Yichen Ma 3 4,  Jinying Peng 1,  Xiu-Jie Wang 5 6,  Chengqi Yi 7 8 9 10
Affiliations

Affiliations

  • 1 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
  • 2 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
  • 3 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
  • 4 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
  • 5 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China. xjwang@genetics.ac.cn.
  • 6 School of Future Technology, University of Chinese Academy of Sciences, Beijing, China. xjwang@genetics.ac.cn.
  • 7 State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  • 8 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  • 9 Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  • 10 Beijing Advanced Center of RNA Biology (BEACON), Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  • # Contributed equally.
PMID: 40325216 DOI: 10.1038/s41592-025-02680-9
Abstract

Methods for absolute quantification of N6-methyladenosine (m6A) have emerged as powerful tools in epitranscriptomics. We previously reported GLORI, a chemical-assisted approach to achieve unbiased and precise m6A measurement. However, its lengthy reaction time and severe RNA degradation have limited its applicability, particularly for low-input samples. Here, we present two updated GLORI approaches that are ultrafast, mild and enable absolute m6A quantification from one to two orders of magnitude less than the RNA starting material: GLORI 2.0 is compatible with RNA from ~10,000 cells and enhances sensitivity for both transcriptome-wide and locus-specific m6A detection; GLORI 3.0 further utilizes a reverse transcription-silent carrier RNA to achieve m6A quantification from as low as 500-1,000 cells. Using limited RNA from mouse dorsal hippocampus, we reveal a high modification level in synapse-related gene sets. We envision that the updated GLORI methods will greatly expand the applicability of absolute quantification of m6A in biology.

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