SAC-seq for transcriptome-wide quantitative sequencing of mRNA m6A at base resolution in Arabidopsis

Here, we reported transcriptome-wide m6A modification maps within single-base resolution using m6A-SAC-seq in rice and Arabidopsis. Our analysis uncovered a total of 205,691 m6A sites distributed across 22,574 genes in rice, and 188,282 m6A sites across 19,984 genes in Arabidopsis. Overall design: To establish a comprehensive atlas to investigate the tissue specificity and evolutionary conservation of poly(A) RNA m6A regulation in plants, we first extracted the total RNA of nine Arabidopsis tissues (seedling, shoot, root, rosetta leaf, cauline leaf, stem, flower, silique and seed) as well as eight rice tissues (plumule dark, plumule light, Seedling at 8 days, Seedling at 2 weeks, panicle, flag leaf at 10 days after anthesis, endosperm at 10 days after anthesis, and embryo at 10 days after anthesis with two biological replicates for each sample (Fig. 1b, c ). poly(A) RNA of each biological replicate was captured first for LC-MS/MS to measure the m6A/A ration in the whole transcriptome.

本研究利用m6A-SAC-seq技术，构建了水稻与拟南芥的单碱基分辨率全转录组N6-甲基腺嘌呤（m6A）修饰图谱。经分析，水稻中共鉴定到分布于22574个基因上的205691个m6A修饰位点，拟南芥中共鉴定到分布于19984个基因上的188282个m6A修饰位点。总体实验设计：为构建全面的图谱以探究植物聚腺苷酸化RNA（poly(A) RNA）的m6A调控的组织特异性与进化保守性，我们首先提取了9种拟南芥组织（幼苗、地上部、根、莲座叶、茎生叶、茎、花、角果与种子）以及8种水稻组织（黑暗培养胚芽、光照培养胚芽、8天龄幼苗、2周龄幼苗、穗、开花后10天的旗叶、开花后10天的胚乳、开花后10天的胚），所有样品均设置2个生物学重复（图1b、c）。针对每个生物学重复样品，我们首先富集其聚腺苷酸化RNA，随后通过液相色谱-串联质谱（LC-MS/MS）检测全转录组范围内的m6A/A比值。