title: High-Resolution Mapping of N⁶-Methyladenosine in Transcriptome and Genome Using a Photo-Crosslinking-Assisted Strategy.
abstract: A photo-crosslinking-assisted strategy has been developed and applied to the high-resolution mapping of N(6)-methylation of adenosine (N(6)-methyladenosine or m(6)A/6mA, we chose to use m(6)A to represent the RNA methylation, and 6mA to represent the DNA methylation in order to be consistent with DNA 5mC methylation nomenclature) in transcriptome and genome. The new approach introduces a covalent interaction between the anti-m(6)A antibody and the RNA/DNA molecule, followed by multiple washing steps which reduce nonspecific binding in immunoprecipitation and nuclease digestion in order to significantly increase the resolution by removing unprotected polynucleotides. By using this protocol, a high-resolution transcriptome-wide human m(6)A map and genome-wide Chlamydomonas 6mA map were obtained, providing new insights into the distribution and biological functions of the m(6)A/6mA. This well-established strategy is reproducible and widely applicable to other biological systems for high-throughput sequencing investigations of other RNA/DNA modifications.
authors.affiliation: Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA; Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA; Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois, USA.
authors.forename: Guan-Zheng
authors.initials: GZ
authors.lastname: Luo
authors.affiliation: Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois, USA; Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois, USA. Electronic address: chuanhe@uchicago.edu.
authors.forename: Chuan
authors.initials: C
authors.lastname: He
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D002874:Chromosome Mapping / Q000379:methods*; D019175:DNA Methylation / Q000235:genetics; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D059467:Transcriptome / Q000235:genetics*
chemical_list: D000888:Antibodies, Anti-Idiotypic; C010223:N-methyladenosine; D000241:Adenosine
keywords: High-throughput sequencing; N(6)-Methyladenosine; Nuclease digestion; Photo-crosslinking