m6A-seq of the mouse ESC transcriptome expressing wild-type (R-WT) or non-phosphorylatable (R-3A2A) METTL3/WTAP

We performed immunoprecipitation of m6A-modfiied poly-A tailed RNA in mouse ESCs expressing either wild-type or non-phosphorylatable METTL3/WTAP. By sequencing over 4 billion bases across both Input and Immunoprecipitated (IP) samples, we generate a transcriptome-wide map of m6A methylation enrichment across both cell types. We find that m6A peaks that are differentially methylated between R-WT and R-3A2A mouse ESCs. Several pluripotency transcripts show reduced m6A methylation in R-3A2A cells compared to R-WT. For the CRISPR screen, we transduced a circular RNA GGACU-containing GFP reporter plasmid, followed by transduction of a genome-wide CRISPR sgRNA library. We selected for the top and bottom 5% of GFP expression in HeLa cells and sequenced the sgRNAs. Overall design: Profiles of m6A methylation on mRNA of wild type (WT) and mutant (3A2A) mouse ESCs were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000. CRISPR sgRNA sequencing was performed on HeLa cells expressing the highest and lowest 5% of GFP signal.