RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was highly efficient, resulting in less than 1.5% rRNA content in the final library, significantly better than other reported rRNA removal techniques. We identified >30,000 unique transcripts from all samples, including protein-coding genes and many unique species of non-coding RNA, in biologically-relevant proportions. Furthermore, normalized sequencing read count for select genes significantly negatively correlated with Ct values from RT-qPCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

中文翻译：

---

通过缺失核糖体RNA的RNA-Seq同时检测编码和非编码转录本的工作流程。

RNA测序为转录组提供了前所未有的途径。关键在于同时鉴定和定量同一样品中许多不同种类的RNA。在这项研究中，我们描述了一种新的协议，用于同时检测离子总RNA-Seq试剂盒v2协议并结合QIASeq FastSelect rRNA去除试剂盒，同时检测编码和非编码转录本。我们报告高度一致的测序文库可以从冷冻的高完整性小鼠海马组织和更具挑战性的验尸后的人类组织中产生。使用FastSelect去除rRNA的效率很高，导致最终文库中的rRNA含量不到1.5％，明显优于其他报道的rRNA去除技术。我们从所有样本中识别出超过30,000个独特的成绩单，包括与蛋白质相关的比例的蛋白质编码基因和许多非编码RNA的独特物种。此外，来自相同样本的RT-qPCR分析中所选基因的标准化测序读取计数与Ct值显着负相关。这些结果表明，该协议可同时准确，一致地识别和量化各种转录本。高效的rRNA消耗，加上最小化的样品处理，并且没有复杂且高损失的大小选择方案，使得该方案对希望研究整个转录组的研究人员很有用。选定基因的标准化测序读取计数与相同样品的RT-qPCR分析的Ct值显着负相关。这些结果表明，该协议可同时准确，一致地识别和量化各种转录本。高效的rRNA消耗，加上最小化的样品处理，并且没有复杂且高损失的大小选择方案，使得该方案对希望研究整个转录组的研究人员很有用。选定基因的标准化测序读取计数与相同样品的RT-qPCR分析的Ct值显着负相关。这些结果表明，该协议可同时准确，一致地识别和量化各种转录本。高效的rRNA消耗，加上最小化的样品处理，并且没有复杂且高损失的大小选择方案，使得该方案对希望研究整个转录组的研究人员很有用。