title: Mapping m6A at Individual-Nucleotide Resolution Using Crosslinking and Immunoprecipitation (miCLIP).
abstract: N6-methyladenosine (m6A) is the most abundant modified base in eukaryotic mRNA and has been linked to diverse effects on mRNA fate. Current m6A mapping approaches localize m6A residues to 100-200 nt-long regions of transcripts. The precise position of m6A in mRNAs cannot be identified on a transcriptome-wide level because there are no chemical methods to distinguish between m6A and adenosine. Here, we describe a method for using anti-m6A antibodies to induce specific mutational signatures at m6A residues after ultraviolet light-induced antibody-RNA crosslinking and reverse transcription. Then, we describe how to use these mutational signatures to map m6A residues at nucleotide resolution. Taken together, our protocol allows for high-throughput detection of individual m6A residues throughout the transcriptome.
authors.affiliation: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, 10065, USA.
authors.forename: Anya V
authors.initials: AV
authors.lastname: Grozhik
authors.affiliation: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, 10065, USA.
authors.forename: Bastian
authors.initials: B
authors.lastname: Linder
authors.affiliation: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, 10065, USA.
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Olarerin-George
authors.affiliation: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, 10065, USA.
authors.forename: Samie R
authors.initials: SR
authors.lastname: Jaffrey
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives*; D000906:Antibodies; D019295:Computational Biology / Q000379:methods; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D047468:Immunoprecipitation / Q000379:methods; D007553:Isotope Labeling; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012984:Software
chemical_list: D000906:Antibodies; D012313:RNA; C010223:N-methyladenosine; D000241:Adenosine
keywords: Crosslinking; High-throughput sequencing; N 6 -Methyladenosine; RNA