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

Authors :
Dierks D
Garcia-Campos MA
Uzonyi A
Safra M
Edelheit S
Rossi A
Sideri T
Varier RA
Brandis A
Stelzer Y
van Werven F
Scherz-Shouval R
Schwartz S
Source :
Nature methods [Nat Methods] 2021 Sep; Vol. 18 (9), pp. 1060-1067. Date of Electronic Publication: 2021 Sep 03.
Publication Year :
2021

Abstract

N <superscript>6</superscript> -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.<br /> (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Details

Language :
English
ISSN :
1548-7105
Volume :
18
Issue :
9
Database :
MEDLINE
Journal :
Nature methods
Publication Type :
Academic Journal
Accession number :
34480159
Full Text :
https://doi.org/10.1038/s41592-021-01242-z