The effects of a single bout of exercise on the transcriptome and m6A modification in mouse iWAT

Mice were subjected to a single 1-hour exercise session, followed by a 4-hour rest period. iWAT was collected, and RNA was extracted. The RNA was used for RNA-seq and m6A methylation-seq (MeRIP-seq). MeRIP-seq was carried out based on the previously reported protocol, with minor modifications. In brief, 100 μg total RNA was used for mRNA purification by the Oligo (dt) Dynabeads (Thermo Fisher Scientific, 61012). Subsequently, a non-contact ultrasonic disruptor (Diagenode, Bioruptor Pico) was used to sonicate 2 μg of mRNA for 30 cycles, fragmenting it into approximately 150-200 nt fragments. Approximately 1/10 of the fragmented RNA was saved as the input control for further RNA-seq by RNA-seq Library Prep Kit (Vazyme, NR604). The Protein G Magnetic Beads (NEB, S1430) were incubated with anti-m6A antibody (NEB, E1610S) at 4℃ for 1 hour with rotation, followed by washing off unbound antibody and resuspension in immunoprecipitation buffer. The remaining 9/10 of the RNA was mixed with the aforementioned suspension and incubated at 4℃ with rotation for 4 hours. RLT buffer (Qiagen, 79216) was added, and the solution was incubated at room temperature for 10 minutes to dissociate the methylated RNA obtained from MeRIP for RNA-seq library construction. For both the Input and IP libraries, we commissioned GENEWIZ (Suzhou, China) to sequence each sample on the Illumina Novaseq 6000, PE150 platform with 8G per sample.