Summary

• 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.

Abstract

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an 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, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution