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.小鼠接受单次1小时运动干预，随后静置休息4小时。采集其腹股沟白色脂肪组织（inguinal white adipose tissue, iWAT）并提取总RNA。所提取的RNA分别用于RNA测序（RNA-seq）和m⁶A甲基化测序（m⁶A methylation-seq，即MeRIP-seq）。MeRIP-seq实验基于已发表的实验方案开展，仅做了小幅优化。简言之，取100 μg总RNA，采用寡聚(dT)磁珠（Oligo (dt) Dynabeads，赛默飞世尔科技，货号61012）进行mRNA纯化。随后，取2 μg纯化后的mRNA，使用非接触式超声破碎仪（Diagenode，Bioruptor Pico）进行30个循环的超声打断，将mRNA片段化至约150~200 nt的长度。取约1/10的片段化RNA作为输入对照，使用RNA-seq文库制备试剂盒（诺唯赞，货号NR604）进行后续RNA-seq建库。将蛋白G磁珠（Protein G Magnetic Beads，新英格兰生物实验室，货号S1430）与抗m⁶A抗体（NEB，货号E1610S）于4℃条件下旋转孵育1小时，随后洗脱未结合的抗体，并重悬于免疫沉淀缓冲液中。将剩余9/10的片段化RNA与上述磁珠-抗体悬液混合，于4℃条件下旋转孵育4小时。加入RLT缓冲液（凯杰，货号79216），室温孵育10分钟以解离免疫沉淀得到的甲基化RNA，用于后续RNA-seq文库构建。对于输入对照文库与免疫沉淀（IP）文库，我们委托中国苏州的金唯智（GENEWIZ）在Illumina NovaSeq 6000 PE150测序平台上对每个样本进行测序，每个样本的测序数据量为8G。