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

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.