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

单细胞高精度小 RNA 测序 (RealSeq-SC)

基本信息

批准号：
9406996
负责人：
Sergio Barberan-Soler
金额：
$ 28.12万
依托单位：
SOMAGENICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-10 至 2019-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9406996
关键词：
Adverse effects Affect Applications Grants Automation Biotechnology Breast Cancer cell line Buffers Cells Chemicals Complementary DNA Coupled Cytolysis Detection Development Digestion Dimerization Evolution Excision Gene Expression Gene Expression Regulation Generations Genetic Transcription Goals Health Heterogeneity High-Throughput Nucleotide Sequencing Homeostasis Individual Libraries Ligation Liquid substance Malignant Neoplasms Measures Mediating Messenger RNA Methods MicroRNAs Modernization Modification Molecular Monitor Noise Oligonucleotides Phase Play Population Preparation Protocols documentation RNA RNA library Reaction Reporting Research Reverse Transcription Ribonuclease H Ribosomal RNA Role Sampling Scheme Small Business Innovation Research Grant Small RNA Splint Device System Technology Testing Transcript Tube base circular RNA design dimer innovation neoplastic cell next generation sequencing novel strategies pre-miRNA repaired single cell analysis single cell sequencing single cell technology transcriptome transcriptomics tumor progression

项目摘要

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.

抽象的该拨款申请的目标是开发第一个用于分析的商用文库制备试剂盒使用 NGS 方法对单细胞中的小 RNA 进行单细胞分析已成为可能。这些发现鉴定了原本被认为相同的细胞之间的关键差异。研究表明，基因表达调控中存在内在的“噪音”，在不幸的是，目前缺乏关于细胞间变异的信息。事实上，没有市售的文库制备试剂盒。对于可以分析单细胞的小RNA，我们建议进一步开发我们的文库制备技术， RealSeq®-AC 能够定量单细胞中的小 RNA，其使用的方案涉及单个组合适配器和环化可准确定量超过 75% 的检测到的 miRNA，与最佳竞争对手套件的约 35% 相比，为了将该技术应用于单细胞测序，我们将进行测试。三种不同的策略可显着减少文库中接头二聚体的存在，以及我们将开发一种可能使用 miRNA 预扩增来减少所需 PCR 循环数的方法。在单管中执行从细胞裂解到扩增文库最终纯化的所有步骤的方案。技术将能够准确定量单细胞中的小RNA。