Detection of Bidirectional Promoter-Derived lncRNAs from Small-Scale Samples Using Pre-Amplification-Free Directional RNA-seq Method
Detection of Bidirectional Promoter-Derived lncRNAs from Small-Scale Samples Using Pre-Amplification-Free Directional RNA-seq Method
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使用无预扩增定向 RNA-seq 方法检测小规模样品中双向启动子衍生的 lncRNA
DOI：
10.1007/978-1-4939-6988-3_6
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发表时间：
2017
影响因子：
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通讯作者：
Imamura Takuya
中科院分区：
文献类型：
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作者：
Hamazaki Nobuhiko;Nakashima Kinichi;Hayashi Katsuhiko;Imamura Takuya
高通量测序技术的发展揭示了长链非编码RNA （lncRNA）的广阔世界。来自双向基因启动子的分散转录lncrna被称为启动子相关非编码rna (promoter-associated noncoding RNAs, pancRNAs)，约占lncrna总数的20%，这一主要部分参与了许多生物学过程，如发育和癌症形成。最近，我们发现胰腺rna可以激活它们的伴侣基因，如在合子基因激活（ZGA）开始时由白细胞介素17d （Il17d）启动子区域反义链转录的胰腺rna pancil17d，通过il17双调控对小鼠着床前发育至关重要。通过使用一种从小规模样本中生成定向RNA-seq文库的方法，在ZGA期间发现了一组特定的pancrna的表达。虽然有几种方法可用于小规模样本，但大多数方法都需要预扩增程序，这通常会对转录本子集产生一些扩增偏差。我们在此提供了一种高灵敏度和可重复性的方法，该方法基于制备方向性RNA-seq文库，仅需100个小鼠卵母细胞或胚胎，无需预扩增即可用于lncrna和mrna的定量。
Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact thatpancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region ofInterleukin 17d (Il17d)at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development throughIl17dupregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs.