MeRIP-seq for m6A Transcriptome Mapping

The MeRIP-seq technique was developed on the basis of published experimental methods 18 (link)]. Briefly, fragmented RNA was incubated with anti-m⁶A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer for 2 hours at 4°C. The reaction mixture was further immunoprecipitated with Protein A magnetic beads (Thermo Fisher) at 4°C for 2 hours. Then, bound RNA was eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted with Trizol reagent (Thermo Fisher) according to the manufacturer’s instruction. Purified RNA was used for RNA-seq library generation with the NEBNext® Ultra™ RNA Library Prep Kit (NEB). Both the input sample without immunoprecipitation and the m⁶A IP samples were subjected to 150 bp paired-end sequencing on Illumina HiSeq sequencer.