Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Equipe Labellisée Ligue Contre le Cancer, Centre National de la Recherche Scientifique UMR 7104, Institut National de la Santé et de la Recherche Médicale U964, University of Strasbourg, Illkirch, France.
Nat Protoc. 2015 Dec;10(12):1915-38. doi: 10.1038/nprot.2015.120. Epub 2015 Oct 29.
High-throughput transcriptional analysis has unveiled a myriad of novel RNAs. However, technical constraints in RNA sequencing library preparation and platform performance hamper the identification of rare transcripts contained within the RNA repertoire. Herein we present targeted-RNA directional sequencing (TARDIS), a hybridization-based method that allows subsets of RNAs contained within the transcriptome to be interrogated independently of transcript length, function, the presence or absence of poly-A tracts, or the mechanism of biogenesis. TARDIS is a modular protocol that is subdivided into four main phases, including the generation of random DNA traps covering the region of interest, purification of input RNA material, DNA trap-based RNA capture, and finally RNA-sequencing library construction. Importantly, coupling RNA capture to strand-specific RNA sequencing enables robust identification and reconstruction of novel transcripts, the definition of sense and antisense RNA pairs and, by the concomitant analysis of long and natural small RNA pools, it allows the user to infer potential precursor-product relations. TARDIS takes ∼10 d to implement.
高通量转录组分析揭示了大量新的 RNA。然而，RNA 测序文库制备和平台性能的技术限制阻碍了对 RNA 谱中包含的稀有转录本的鉴定。在此，我们提出了靶向 RNA 定向测序（TARDIS），这是一种基于杂交的方法，可以独立于转录本长度、功能、是否存在 poly-A 片段或生物发生机制来检测转录组中包含的 RNA 子集。TARDIS 是一个模块化的方案，分为四个主要阶段，包括生成覆盖感兴趣区域的随机 DNA 陷阱、输入 RNA 材料的纯化、基于 DNA 陷阱的 RNA 捕获，最后是 RNA 测序文库构建。重要的是，将 RNA 捕获与链特异性 RNA 测序相结合，可实现新型转录本的稳健鉴定和重建，定义正义和反义 RNA 对，并且通过对长链和天然小 RNA 池的同时分析，可推断潜在的前体-产物关系。TARDIS 大约需要 10 天的时间来实施。
