The effects of ALKBH5 on m6A mRNA modification in the liver [MeRIP-seq]

Through m6A mRNA-profiling, we aim to characterize the m6A mRNA changes in the liver between Alkbh5flox/flox and Alkbh5-HKO mice. Overall design: To map the mRNA m6A modification caused by ALKBH5 in the liver, MeRIP-seq was performed in the livers of Alkbh5flox/flox and Alkbh5-HKO mice. Total RNA was extracted using Tripure Isolation Reagent (Roche, Mannheim, Germany) from livers of Alkbh5flox/flox and Alkbh5-HKO mice at 9 weeks old. A total of 300 µg RNAs were pooled from three mice.Fragmented mRNA (~100 nt) was incubated for 2 hr at 4oC with anti-m6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated mRNAs or Input was used for library construction with NEBNext ultra RNA library prepare kit for Illumina (New England Biolabs). The library preparations were sequenced on an Illumina Novaseq6000 platform with a paired-end read length of 150 bp according to the standard protocols. After mapping reads to the reference genome, exomePeak R package (version 2.16.0) was used for the m6A peak identification in each anti-m6A immunoprecipitation group with the corresponding input samples serving as a control, and q-value threshold of enrichment of 0.05 was used for all data sets. The m6A-enriched motifs of each group were identified by HOMER (version 4.9.1). The liver mRNA m6A profiles in Alkbh5flox/flox and Alkbh5-HKO mice were characterized.

本研究借助N6-甲基腺嘌呤（m6A）信使RNA（mRNA）谱分析技术，旨在解析Alkbh5 flox/flox与Alkbh5-HKO小鼠肝脏组织内mRNA的m6A修饰差异。整体实验设计：为绘制肝脏中ALKBH5介导的mRNA m6A修饰图谱，我们对9周龄Alkbh5 flox/flox与Alkbh5-HKO小鼠的肝脏组织开展甲基化RNA免疫沉淀测序（MeRIP-seq）。我们使用Tripure总RNA提取试剂（罗氏，曼海姆，德国），从上述两种小鼠的肝脏组织中提取总RNA，并将3只小鼠的RNA混合，最终获得总计300微克的RNA样本。将片段化至约100 nt的mRNA与抗m6A多克隆抗体（Synaptic Systems公司）在4℃条件下孵育2小时，用于免疫沉淀实验。随后，将免疫沉淀得到的mRNA以及Input对照样本，采用适配Illumina平台的NEBNext超高效RNA文库制备试剂盒（新英格兰生物实验室，New England Biolabs）完成文库构建。按照标准实验流程，将构建好的文库在Illumina NovaSeq6000测序平台上进行双端150 bp的测序。将测序读段比对至参考基因组后，使用exomePeak R软件包（版本2.16.0），以对应的Input样本作为对照，对每个抗m6A免疫沉淀组进行m6A峰识别，并对所有数据集设置0.05的富集q值作为筛选阈值。采用HOMER软件（版本4.9.1）识别各组的m6A富集基序。本研究最终完成了Alkbh5 flox/flox与Alkbh5-HKO小鼠肝脏组织mRNA m6A谱的特征解析。