GSM8242660: db3 Input; Mus musculus; RNA-Seq
Total RNAs were obtained from db/db mice and lean control mice using TRIzol reagent (catalog T9108, Takara) according to the manufacturer's protocol. Fragmented RNA was incubated with anti-m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer for 2 hours at 4°C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 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) by following the manufacturer's instruction. Purified RNA was used for RNA-seq library generation with 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.