RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs. RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. Overall design: A single RNA sample was divided into 6 technical replicates. Three of these technical replicates were treated with Tobacco Acid Phosphatase (TAP, Epicentre) and the other three, with RNA 5’Pyrophosphohydrolase (RppH, New England Biolabs, #M0356S).

RNA干扰（RNA interference）首次在秀丽隐杆线虫（Caenorhabditis elegans）中被描述。自此之后，已有多种新型内源小RNA通路被不同程度地报道并表征。与植物类似，但与果蝇（Drosophila）和哺乳动物不同，线虫拥有RNA依赖的RNA聚合酶（RNA-dependent RNA Polymerases, RdRPs），可利用其他转录本作为模板直接合成小RNA。秀丽隐杆线虫中由RdRPs RRF-1和EGO-1产生的一类极具代表性的次级小干扰RNA（亦被称为22G-RNAs），即便在装载进入Argonaute蛋白后，仍保留了源自RdRP催化活性的5’三磷酸基团。这带来了一项技术难题：5’PPP基团会降低小RNA测序的克隆效率。为提升这类小RNA分子的克隆效率，该领域的常规操作是在文库构建前，使用烟草酸焦磷酸酶（Tobacco Acid pyrophosphatase, TAP）处理RNA样本。近期，烟草酸焦磷酸酶的生产与供应已终止，因此亟需开发替代方案。我们转而选用了从大肠杆菌（E. coli）中分离得到的商业化焦磷酸酶——RNA 5’焦磷酸水解酶（RNA 5’ pyrophosphohydrolase, RppH）。本研究直接对比了TAP与RppH在小RNA文库构建中的应用效果。实验整体设计：将单份RNA样品分为6个技术重复样本，其中3份经烟草酸磷酸酶（Tobacco Acid Phosphatase, TAP, Epicentre）处理，剩余3份经RNA 5’Pyrophosphohydrolase（RppH, New England Biolabs, #M0356S）处理。