RNA methylation in diabetes [RNA-Seq]

The MeRIP-seq was carried out in Novogene (Beijing, China). Briefly, 2 μg total RNA was extracted from the retinas of both wide type mice and their littermates with diabetes. The integrity and concentration of extracted RNA was detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare), respectively. Fragmented RNA (~100 nt) was incubated for 2 hours at 4 ℃ with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated RNA or input was used for library construction with Ovation SoLo RNA-Seq System Core Kit (NuGEN). The library preparations were sequenced on Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols. The sequencing was carried out with three independent biological replicates.本实验的甲基化RNA免疫沉淀测序（MeRIP-seq）由位于中国北京的诺禾致源（Novogene）完成。简言之，从野生型（wild type）小鼠及其糖尿病同窝仔鼠的视网膜中提取2 μg总RNA；分别采用安捷伦2100生物分析仪（Agilent）与simpliNano分光光度计（GE Healthcare）检测所提取RNA的完整性与浓度。免疫沉淀实验中，将长度约100 nt的片段化RNA与抗m6A多克隆抗体（Synaptic Systems）于4℃孵育2小时；随后将免疫沉淀获得的RNA或输入对照RNA，使用Ovation SoLo RNA测序系统核心试剂盒（NuGEN）进行文库构建。按照标准实验流程，在Illumina NovaSeq测序平台对构建好的文库进行测序，测序模式为双端150 bp读长；本次测序设置3次独立生物学重复。