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Identification of m⁶A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline.

ABSTRACT: Methylation at the N⁶ position of adenosine (m⁶A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m⁶A nucleotides within transcripts has been historically challenging due to m⁶A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m⁶A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m⁶A residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-m⁶A antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m⁶A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m⁶A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m⁶A sites at true single-nucleotide resolution. As the effort to accurately identify m⁶As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m⁶A epitranscriptome.

SUBMITTER: Roberts JT

PROVIDER: S-EPMC7962486 | biostudies-literature |

REPOSITORIES: biostudies-literature

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