N6-methyladenosine (m6A) is the most prevalent modification of mRNA in mammals and plays a major role in the post-transcriptional regulation of gene expression. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels of granularity: The modified site, the overall m6A levels per gene, and the global levels per sample. Current approaches address these needs in a highly limited manner. Here we develop m6A-seq2, relying on multiplexed m6A immunoprecipitation of pre-barcoded and pooled samples. m6A-seq2 allows a major increase in throughput, while dramatically reducing technical variability, requirements of input material, cost, and labor. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an important, 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 in terms of m6A levels. This is ~10 fold higher than could be observed using previous approaches and establishes m6A as a major driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-based regulation at three critical resolutions. Overall design: polyA-selected SK1 RNA. 12 technical replicates for m6A-seq2 analysis, 3 technical replicates for m6A-seq. 4 batches of m6A-seq2 analysis of various genetic perturbations in SK1 Ndt-/- background. Yeast meiosis timecourse 12 samples (5 timepoints, duplicates) and a positive and negative control prepared with m6A-seq2 protocol. 5 clones with m6A-'writer' genetic perturbation (partial knock-out), 2 Wtap and 2 Mettl3 perturbations in mouse embryonic stem cells prepared with m6A-seq2 protocol. 24 samples poly-A selected samples (serial dilution of WT and Mettl3 KO mESC RNA in technical triplicates) conducted to m6A-seq2. 10 technical replicates of MEF poly-selected RNA
