Detection of m6A RNA modifications at single-nucleotide resolution using m6A-selective allyl chemical labeling and sequencing

Yong Peng; Hanzhe Meng; Ruiqi Ge; Shun Liu; Mengjie Chen; Chuan He; Lulu Hu

doi:10.1016/j.xpro.2022.101677

Detection of m⁶A RNA modifications at single-nucleotide resolution using m⁶A-selective allyl chemical labeling and sequencing

STAR Protoc. 2022 Dec 16;3(4):101677. doi: 10.1016/j.xpro.2022.101677. Epub 2022 Sep 15.

Authors

Yong Peng¹, Hanzhe Meng², Ruiqi Ge³, Shun Liu¹, Mengjie Chen⁴, Chuan He⁵, Lulu Hu⁶

Affiliations

¹ Department of Chemistry, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA; Section of Genetic Medicine, Department of Medicine, Department of Human Genetics, The University of Chicago, Chicago, IL, USA.
² Fudan University Institutes of Biomedical Sciences, Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Shanghai Medical College of Fudan University, Shanghai 200032, China.
³ Department of Chemistry, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA.
⁴ Section of Genetic Medicine, Department of Medicine, Department of Human Genetics, The University of Chicago, Chicago, IL, USA.
⁵ Department of Chemistry, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA. Electronic address: [email protected].
⁶ Fudan University Institutes of Biomedical Sciences, Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Shanghai Medical College of Fudan University, Shanghai 200032, China. Electronic address: [email protected].

Abstract

As the most abundant internal mRNA modification, N⁶-methyladenosine (m⁶A) was involved in almost all the aspects of RNA metabolism. Here, we introduce our protocol for m⁶A-SAC-seq, which enables the whole transcriptome-wide mapping of m⁶A RNA modification at single-nucleotide resolution with stoichiometry information. m⁶A-SAC-seq relies on selective allyl labeling of m⁶A by specific methyltransferase and chemical treatment that introduce mutation upon reverse transcription. The technique only requires ∼30 ng of input RNA. For complete details on the use and execution of this protocol, please refer to Hu et al. (2022).

Keywords: Bioinformatics; Biotechnology and bioengineering; Chemistry; Genomics; Molecular biology; Molecular/Chemical probes; Protein biochemistry; Protein expression and purification; Sequence analysis.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Adenosine* / genetics
Methylation
Nucleotides*
RNA / genetics
Transcriptome

Substances

Nucleotides
Adenosine
RNA

Abstract

Publication types

MeSH terms

Substances

Grants and funding