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Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.
- 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.)
- Subjects :
- Adenosine analysis
Adenosine genetics
Animals
Cell Cycle Proteins genetics
Cell Cycle Proteins metabolism
Gene Expression
Half-Life
Meiosis
Methyltransferases genetics
Methyltransferases metabolism
Mice
Mice, Knockout
Mouse Embryonic Stem Cells cytology
Mouse Embryonic Stem Cells physiology
RNA Splicing Factors genetics
RNA Splicing Factors metabolism
Yeasts genetics
Adenosine analogs & derivatives
RNA Stability genetics
Sequence Analysis, RNA methods
Subjects
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