An antibody independent verification method quantifies m6A in the chicken ß-actin zipcode

N6-Methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of the high throughput detection of methylated sites in mRNA using MeRIPSeq or miCLIP revolutionized the m6A research field. Both methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. Currently such approaches are limited. Here we present RedBaron, an improved biochemical method for the site-specific detection and quantification of m6A in RNA. We demonstrate that the RedBaron method is able to accurately quantify m6A levels at specific transcripts in vivo. We used this assay for the site-specific detection and quantification of m6A within the chicken ß-actin (ACTB) zipcode sequence in chicken embryos and in fibroblast cells. We demonstrate that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, whilst methylation of a nearby adenosine abolishes Overall design: Fragmented poly(A) RNA from 3 replicates of chicken embryos stage Hamburger-Hamilton 27 was immunoprecipitated using anti-m6A-antibody from NEB, and subjected to next generation sequencing. The sequencing results were compared to the RNAseq results from the same samples.