Dec 25, 2025

Public workspaceFunctional mapping of m6A RNA modifications in cancer

Nature Cancer
DOI
https://dx.doi.org/10.17504/protocols.io.5jyl8xnr7v2w/v1
  • Xin Xu1,
  • Yujuan Wang1,
  • Magnus Lam1
  • 1Princess Margaret Cancer Centre
Xin Xu
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8xnr7v2w/v1
External link: https://doi.org/10.1038/s43018-026-01117-2
Protocol Citation: Xin Xu, Yujuan Wang, Magnus Lam 2025. Functional mapping of m6A RNA modifications in cancer. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xnr7v2w/v1
Manuscript citation:
Xin Xu, Yujuan Wang, Helen Zhu, Magnus Lam, Wenqin Luo, Mona Teng, Yin Liu, Wang Yuan Guo, Aastha Aastha, Xi Xu, Sujun Chen, Xinpei Ci, Shiyan Wang, Yong Zeng, Guanghui Zhu, Thomas Kislinger, Mathieu Lupien, Ming-Sound Tsao, Housheng Hansen He (2026) METTL3-based epitranscriptomic editing screening identifies functional m6A sites in cancers.Nature Cancer doi: 10.1038/s43018-026-01117-2
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 21, 2025
Last Modified: December 25, 2025
Protocol Integer ID: 235604
Keywords: functional mapping of m6a rna modification, m6a rna modification, specific m6a validation, including chromatin immunoprecipitation, rna immunoprecipitation, chromatin immunoprecipitation, m6a, cancer this protocol, immunofluorescence assay, cancer, immunofluorescence
Disclaimer
This overview protocol is provided for research use only. Individual experimental procedures may require optimization depending on experimental conditions.
Abstract
This protocol provides an overview of the experimental workflows used in this study, including chromatin immunoprecipitation (ChIP), m6A RNA immunoprecipitation (MeRIP-seq), SELECT-based site-specific m6A validation, and immunofluorescence assays. Detailed step-by-step protocols for each procedure are provided separately and linked below.
Troubleshooting
Before start
  • All experiments were performed using standard molecular biology and cell culture practices.
  • Detailed reagent compositions, incubation conditions, and quality control steps are described in the individual protocols linked below.
  • Users should consult the corresponding protocol for step-by-step instructions and optimization guidance.
Chromatin immunoprecipitation (ChIP)
This workflow describes ChIP experiments performed in 22Rv1 cells with or without CHD9 knockdown, followed by DNA purification and ChIP-seq library preparation.

Detailed protocol: Chromatin immunoprecipitation (ChIP) in 22Rv1 cells
https://www.protocols.io/edit/chromatin-immunoprecipitation-chip-in-22rv1-cells-hibjb4akp
ATAC-seq for chromatin accessibility profiling
This workflow describesan assay for transposase-accessible chromatin using sequencing (ATAC-seq) to profile chromatin accessibility in cultured cells.

Detailed protocol: ATAC-seq library preparation in cultured cells https://www.protocols.io/edit/atac-seq-library-preparation-in-cultured-cells-hib7b4arp
m6A RNA immunoprecipitation (MeRIP)
This workflow describes m6A RNA immunoprecipitation using fragmented total RNA from cell lines, followed by purification of m6A-enriched RNA for downstream assays.

Detailed protocol: m6A methylated RNA immunoprecipitation (MeRIP) in cell lines https://www.protocols.io/edit/m6a-merip-hibsb4anf
Senescence-associated β-galactosidase staining
This workflow describes senescence-associated β-galactosidase staining assays used to assess cellular senescence by light microscopy.

Detailed protocol: Senescence β-galactosidase staining assay in cultured cells
https://www.protocols.io/edit/senescence-galactosidase-staining-assay-in-culture-hib5b4aq7
SELECT-based site-specific m6A validation
This workflow describes SELECT (Single-base Elongation- and Ligation-based qPCR Amplification) assays used for site-specific validation of m6A modifications at defined RNA loci.

Detailed protocol: SELECT assay for site-specific m6A detection https://www.protocols.io/edit/select-based-quantification-of-site-specific-m6a-m-hibxb4app
Immunofluorescence staining and imaging
This workflow describes immunofluorescence staining and imaging procedures used to assess protein localization and subcellular distribution in cultured cells.

Detailed protocol: Immunofluorescence staining and imaging in cultured cells https://www.protocols.io/edit/immunofluorescence-staining-and-confocal-microscop-hib4b4aqx