m6A Immunoprecipitation and RNA Sequencing

Fragmented RNA was incubated with an anti-m⁶A polyclonal antibody (Synaptic Systems, 202003) in immunoprecipitation (IPP) buffer for 2 h at 4 °C^{25 (link)}. The reaction mixture was then immunoprecipitated with protein A magnetic beads (Thermo Fisher, MA, USA) at 4 °C for 2 h. Next, the bound RNA was eluted from the beads with N6-methyladenosine antibody in IPP buffer and extracted with TRIzol reagent (Thermo Fisher, MA, USA). The extracted RNA was then prepared with a NEBNext Ultra II Directional RNA Library Prep Kit (NEB, MA, USA). Both the input sample (without immunoprecipitation) and the m⁶A immunoprecipitational samples were subjected to 150 bp paired-end sequencing on an Illumina HiSeq sequencer.