Map RNA Methylation Sites Transcriptome-Wide

m6A-eCLIP

High resolution maps of RNA Methylation

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Technology Overview

m6A-eCLIP™, also known as methylated RNA crosslinking and immunoprecipitation, is a cutting-edge technique allowing researchers to identify m6A-modifications, transcriptome-wide, with single nucleotide resolution, thus providing the ability to elucidate the functional consequences of m6A in gene regulation.

N6-methyladenosine (m6A) is the most prevalent RNA modification found in eukaryotes. m6A modification has been shown to regulate many aspects of RNA biology including splicing, secondary and tertiary structure, nuclear export, localization, stability, and translation. Levels of m6A modification at specific positions is tuned by writer and eraser enzymes and has been shown to play a role in stem cell renewal and differentiation, and dysregulation of m6A states has been implicated in a wide variety of cancers

m6A-eCLIP enables researchers to perform integrative analyses with other genomic data such as RBP-eCLIP to gain a comprehensive understanding of RNA regulatory mechanisms. The information obtained from m6A-eCLIP studies contributes to the development of targeted therapies and the discovery of biomarkers for diseases such as cancer, neurological disorders, and metabolic disorders.

Technology Highlights

Technology Highlights

Unbiased m6A Modification Site Specificity

  • Consistent with the known feature of m6A modifications, peaks of m6A-eCLIP reads are enriched near the stop codons, and for the 5 nt DRACH motif
  • m6A-eCLIP shows similar read densities to a low-resolution m6A profiling technique m6A-RIP (meRIP), but with more precise site calls

Accurate Single Nucleotide Resolution

  • Reverse transcriptase (RT) termination at the crosslink sites of the m6A antibody to RNA form sharp cliffs of aligned read starts enabling single-nucleotide resolution modification calls 
  • Identified sites primarily occur on adenosine bases in a DRACH sequence (the motif preferred by m6A methyltransferases)

High m6A Site Reproducibility

  • High reproducibility for all single nucleotide modification sites between m6A-eCLIP replicates, and even higher reproducibility for modification sites at adenosine in DRACH motifs 
  • Highly correlated single nucleotide modification site enrichment levels in replicate m6A-eCLIP experiments

Unbiased m6A Modification Site Specificity

  • Consistent with the known feature of m6A modifications, peaks of m6A-eCLIP reads are enriched near the stop codons, and for the 5 nt DRACH motif
  • m6A-eCLIP shows similar read densities to a low-resolution m6A profiling technique m6A-RIP (meRIP), but with more precise site calls

Accurate Single Nucleotide Resolution

  • Reverse transcriptase (RT) termination at the crosslink sites of the m6A antibody to RNA form sharp cliffs of aligned read starts enabling single-nucleotide resolution modification calls 
  • Identified sites primarily occur on adenosine bases in a DRACH sequence (the motif preferred by m6A methyltransferases)

High m6A Site Reproducibility

  • High reproducibility for all single nucleotide modification sites between m6A-eCLIP replicates, and even higher reproducibility for modification sites at adenosine in DRACH motifs 
  • Highly correlated single nucleotide modification site enrichment levels in replicate m6A-eCLIP experiments

Publications Citing m6A-eCLIP

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2023

m6A-eCLIP Data Sheet

m6A-eCLIP optimizes the enhanced crosslinking and immunoprecipitation (eCLIP) approach to profile m6A RNA modification sites transcriptome-wide with single nucleotide resolution. m6A-eCLIP requires 100-fold less starting material compared to other m6A profiling methods and yields more precise sites.

2023

m6A-eCLIP Protocol

Starting your m6A-eCLIP Kit? See the full protocol