Identification of m(6)A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Methylation at the N(6) position of adenosine (m(6)A) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific m(6)A nucleotides within transcripts has been historically challenging due to m(6)A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on m(6)A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m(6)A 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-m(6)A 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 m(6)A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m(6)A-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 m(6)A sites at true single-nucleotide resolution. As the effort to accurately identify m(6)As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m(6)A epitranscriptome.