Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution
Published in Nature Methods, 2021
Recommended citation: Dierks, D., Garcia-Campos, M.A., Uzonyi, A., Safra, M., Edelheit, S., Rossi, A., Sideri, T., Varier, R.A., Brandis, A., Stelzer, Y. and van Werven, F., 2021. Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution. Nature Methods, pp.1-8. https://www.nature.com/articles/s41592-021-01242-z
- 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 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.
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