m6A-eCLIP
High Resolution Maps
of the Epitranscriptome
Technology Overview
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 robustly profiles m6A modification sites with single nucleotide resolution transcriptome-wide. In comparison to alternate approaches, m6A-eCLIP requires 100-fold less starting RNA material for m6A profiling. m6A-eCLIP identifies over 70,000 enriched modification sites per sample, of which more than 70% are positioned directly at adenosines and more than 50% are positioned at adenosines in the context of the DRACH motif, a well characterized 5-nucleotide (nt) motif preferred by m6A methyltransferases (the DRACH motif uses IUPAC nucleotide codes where D represents an A, G or U nucleotide, R represents an A or a G nucleotide, and H represents an A, C or U nucleotide).
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
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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
- More than 70% of identified sites are positioned on adenosine nucleotides, and over 50% of all crosslink sites map precisely to adenosines in the DRACH motif preferred by m6A methyltransferases
High m6A Site Reproducibility
- 80% reproducibility for all single nucleotide modification sites between m6A-eCLIP replicates, and even higher reproducibility (83%) for all single nucleotide modification sites at adenosine in DRACH motifs
- High Pearson correlation (> 0.96) for the enrichment ratio of IP reads versus background input reads at individual single nucleotide positions in replicate m6A-eCLIP experiments
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RESOURCES
Services Tailored to Your Experiment
We offer all-inclusive sample processing with expert library preparation and bioinformatic analysis.
