Highly accurate small-RNA sequencing of single cells (RealSeq-SC)

摘要 这项赠款申请的目标是开发第一个可商业化的用于分析的图书馆准备试剂盒 使用NGS方法从单个细胞中提取小RNA。信使核糖核酸的单细胞分析 识别在其他情况下被认为相同的细胞之间的关键差异。这些发现有 研究表明，在基因表达调控中存在着内在的“噪音”，这在 决定细胞的命运。不幸的是，目前缺乏关于细胞间可变性的信息。 ​小RNA的水平，包括microRNA。事实上，没有商业上可用的图书馆准备工具包 用于可以分析单个细胞的小RNA。我们建议进一步发展我们的制库技术， RealSeq®-AC，能够对单个细胞中的小RNA进行量化。RealSeq®-AC使用的方案涉及 单个组合适配器和环形化，准确量化超过75%的检测到的miRNAs， 相比之下，最好的竞争对手套件的比例约为35%。为了使这项技术适用于单细胞测序，我们将测试 三种不同的策略来显著减少文库中的适配二聚体的存在，以及 可能使用miRNA预扩增来减少所需的PCR循环次数。我们将开发一种 一种在单个试管中执行从细胞裂解到最终纯化扩增文库的所有步骤的协议。这 技术将使从单个细胞中准确量化小RNA成为可能。

Abstract The goal of this grant application is to develop the first commercially available library preparation kit for profiling small RNAs from single cells using NGS methods. Single cell analyses of mRNA have allowed the identification of crucial differences between cells that were otherwise considered identical. These findings have shown that there is intrinsic “noise” in the regulation of gene expression that plays an important role in determining cell fates. Unfortunately, there is currently a lack of information about the cell-to-cell variability of levels of ​small RNAs, including microRNAs. Indeed, there is no commercially available library preparation kit for small RNAs that can profile single cells. We propose to further develop our library preparation technology, RealSeq®-AC, to be able to quantify small RNAs from single cells. RealSeq®-AC uses a scheme involving a single combo-adapter and circularization that accurately quantifies over 75% of all miRNAs detected, compared to ~35% from the best competitor kit. To adapt this technology for single-cell sequencing we will test three separate strategies to dramatically reduce the presence of adapter-dimers in the library, as well as possible use of miRNA pre-amplification to reduce the number of PCR cycles needed. We will develop a protocol that performs all steps from cell lysis to final purification of amplified libraries in a single tube. This technology will allow the accurate quantification of small RNAs from single cells.