m6A-seq2 – multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution

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.

Now, researchers at the Weizmann Institute of Science have developed 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, the researchers develop a computational approach for gene-level quantitation of m6A. They 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.

m6A-seq2 reduces batch-induced variability between replicates

a, Schematic comparison of m6A-seq versus m6A-seq2 experimental approach. b, Schematic overview of the m6A quantification approaches on three resolutions (i as index running over k m6A sites; cov for coverage). c, m6A site score comparison between technical replicates generated via m6A-seq (n = 3) or m6A-seq2 (n = 12). Top, heatmap of score 1 for 489 m6A sites. Bottom, score 2 for 577 m6A sites passing the coverage thresholds for high-confidence m6A site estimation. d, Top, comparison of the percentage coefficient of variation (%CV) of score 1 (left) and score 2 (right) estimates across the triplicates of samples measured via m6A-seq, and randomly selected triplicates (from the 12 replicates) measured via m6A-seq2. Boxplot, the center is the median, lower and upper hinges depict the first and third quartile and the whiskers stretch to 1.5 times the interquartile range from the corresponding hinge. Bottom, a histogram of the log₂ fold-change of m6A-seq2 to m6A-seq triplicate CV of the m6A site scores 1 and 2. Mean log₂ fold-change CV indicated with black dashed line. e, Principal component (PC) analysis of score 1 of 486 m6A sites.