- m6A-seq2 is a multiplexed immunoprecipitation-RNA-seq coupled method that measures m6A across the epitranscriptome reducing technical variability, cost, and labor.
- m6A-seq2 allows for sample-level m6A relative quantitation, orthogonal to mass-spectrometry methods, as well as gene-level quantitations.
- m6A-seq2 m6A gene-level measurements explains roughly 30% of the variability in RNA half life on mouse embryonic stem cells.
N6-methyladenosine (m6A) is the most prevalent modification of mRNA in mammals and plays a major role in the post-transcriptional regulation of gene expression. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels of granularity: The modified site, the overall m6A levels per gene, and the global levels per sample. Current approaches address these needs in a highly limited manner. Here we develop m6A-seq2, relying on multiplexed m6A immunoprecipitation of pre-barcoded and pooled samples. m6A-seq2 allows a major increase in throughput, while dramatically reducing technical variability, requirements of input material, cost, and labor. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an important, orthogonal alternative to mass spectrometry based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half-life in mouse embryonic stem cells can be explained in terms of m6A levels. This is ~10 fold higher than could be observed using previous approaches and establishes m6A as a major driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-based regulation at three critical resolutions.