SBIR Phase I: Development of a stand-alone kit for high-throughput and low-cost single-cell RNA sequencing

这个小企业创新研究（SBIR）第一阶段项目的更广泛的影响/商业潜力是开发单细胞RNA测序（scRNA-seq）技术。 单细胞RNA测序已经成为分类细胞类型和了解其功能的最佳工具之一。 scRNA-seq的关键优点是它提供了关于异质样品中单个细胞的转录组范围的信息，而替代方法或者提供关于仅有限的一组基因的信息（例如，流式细胞术，qPCR）或不能提供单细胞分辨率（批量RNA-seq，微阵列）。 然而，目前的scRNA-seq方法仍然昂贵，只能实现有限的样品多路复用，并且与常见的样品储存条件不兼容。该项目将专注于开发满足客户需求的普遍适用的scRNA-seq试剂盒所需的关键技术挑战。如果成功，最终产品将是一个scRNA-seq试剂盒，可以与学术和制药公司客户进行试点测试。通过降低成本，增加scRNA-seq的样本量和样本复用，该技术将提高理解多细胞系统的能力，并增强制药行业的工作流程，如药物筛选和患者分层。SBIR第一阶段项目的智力价值是开发单细胞RNA测序，本发明涉及通过使用标记固定细胞内的转录物的组合条形码方案的scRNA-seq方法。 所提出的技术使得平行测量数十万个单个细胞的转录谱成为可能。与之前的scRNA-seq技术不同，该技术还能够实现高样本复用（高达96个样本/实验），与储存的样本兼容，并在解离前固定细胞，降低细胞处理过程中基因表达扰动的风险。最重要的是，该方法不需要复杂的仪器，这有助于使该技术比现有技术便宜一个数量级。该项目的目标是大幅提高每个细胞检测到的分子数量，并简化用户的工作流程。将测试对分析化学的修改以增加分子检测，并将改变工作流程中耗时的步骤以缩短总体实验时间并降低用户错误的可能性。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to develop technology for single-cell RNA sequencing (scRNA-seq). Single-cell RNA-sequencing has emerged as one of the best tools to catalogue cell types and understand their function. The key advantage of scRNA-seq is that it provides transcriptome-wide information about individual cells in heterogeneous samples while alternative methods either provide information about only a limited set of genes (e.g., flow cytometry, qPCR) or cannot provide single-cell resolution (bulk RNA-seq, microarrays). However, current scRNA-seq approaches remain expensive, enable only limited sample multiplexing, and are not compatible with common sample storage conditions. This project will focus on the key technical challenges that are required to develop universally applicable scRNA-seq kits that meet customer needs. If successful, the end product will be a scRNA-seq kit that is ready for pilot testing with academic and pharmaceutical company customers. By reducing the cost and increasing sample size and sample multiplexing of scRNA-seq, the technology will improve the ability to understand multicellular systems and enhance workflows used in the pharmaceutical industry, such as drug screening and patient stratification.The intellectual merit of this SBIR Phase I project is to develop a single-cell RNA sequencing (scRNA-seq) method by using a combinatorial barcoding scheme that labels transcripts within fixed cells. The proposed technology makes it possible to measure the transcriptional profiles of hundreds of thousands of individual cells in parallel. Unlike previous scRNA-seq technologies, the technology also enables high sample multiplexing (up to 96 samples/experiment), is compatible with stored samples, and fixes cells before dissociation, reducing the risk of perturbations to gene expression during cell handling. Most importantly, the method requires no complex instruments, which contributes to making the technology an order of magnitude less expensive than existing technologies. The objectives of this project are to dramatically improve the number of molecules detected per cell and to simplify the workflow for users. Modifications to assay chemistry will be tested to increase molecular detection, and time-consuming steps in the workflow will be altered to shorten the overall experimental time and reduce the probability of user error.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.