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

David Dierks, Miguel Angel Garcia-Campos, Anna Uzonyi, Modi Safra, Sarit Edelheit, Alice Rossi, Theodora Sideri, Radhika A Varier, Alexander Brandis, Yonatan Stelzer, Folkert van Werven, Ruth Scherz-Shouval, Schraga Schwartz

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)1060-1067
Number of pages8
JournalNature Methods
Volume18
Issue number9
DOIs
StatePublished - 3 Sep 2021

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