Platform for genotyping and phenotyping each cell in a high throughput assay

摘要 一个反复出现的问题，继续激起学者和临床医生的兴趣是了解 罕见细胞的功能、转录和表观遗传程序通常与肿瘤复发有关， 神经系统疾病、慢性感染和其他疾病。大规模并行单细胞基因组工具可以 部分满足了这一市场需求;然而，科学界已经明确表示，数字转录本 计数是不够的，有强大的拉力，提供更全面，多， 一维单细胞数据集。特别地，需要高通量单细胞表型分析工具， 由于使用活细胞的难度增加，其已经落后于基因组学工具的开发。到 为了满足这一需求，Celldom正在开发一种平台，将基于图像的表型分析与单细胞 转录组学在大规模必要的测量活细胞的功能和基因表达， 想要的终点我们通过利用局部图案化的高效微流体阱阵列来实现这种收敛。 打印DNA条形码，这使我们既可以拍摄单细胞的图像，也可以制备条形码scRNA-seq 相同细胞的文库。在我们第一阶段奖励的坚实进展的基础上，我们证明了， 我们的工作流程中的步骤在技术上都是可行的和兼容的，其中包括打印DNA的能力。 密封的微流控芯片内的条形码，捕获阵列中的单个细胞，获取每个细胞的高分辨率图像， 细胞，并最终从附着于表面的条形码引物制备cDNA文库，在此第二阶段，我们将统一 这一工作流程，并展示了在药物发现平台中部署我们的方法的最终目标， 结合了单细胞的延时成像和高分辨率转录组分析。我们的工作计划是 概括为三个具体目标。我们的第一个目标是证明数千个独特的条形码PCR引物， 可以在我们的芯片中以每个腔室格式的一种独特引物打印，并且这些芯片可以用于 制备具有低室交叉污染的高质量scRNA-seq文库。我们的第二个目标是 证明在体外延长时间后，可以成功实施该转录组学工作流程 细胞培养，在此期间，条形码连续暴露于血清中存在的酶和任何 细胞外分泌物我们的第三个目标是证明特定条形码的知识可以使转录本 在合并扩增步骤期间，从特定的腔室中得到选择性富集-这将是 用于证明高优先级克隆的靶向测序，这对我们的目标特别感兴趣。 客户在所有三个目标都被单独展示之后，他们将在决赛中结合起来。 展示了鉴定耐药克隆并探测其转录组特征的能力， 高分辨率。我们希望这次演示能激发潜在客户并吸引他们购买 Celldom仪器用于药物开发应用、治疗和临床诊断。

ABSTRACT One of the recurring questions that continues to pique the interest of academics and clinicians is to understand the functional, transcriptional, and epigenetic programs of rare cells often implicated in tumor recurrence, neurological disorders, chronic infections, and other diseases. Massively parallel single cell genomic tools can partially address this market need; however, the scientific community has made it clear that digital transcript counting is insufficient, and there is strong pull for instruments that provide more comprehensive, multi- dimensional single cell datasets. In particular, there is a need for high-throughput single cell phenotyping tools, which has lagged behind genomics tool development due to the increased difficulty of working with live cells. To meet this demand, Celldom is developing a platform that combines image-based phenotyping with single cell transcriptomics at the massive scales necessary to measure both live cell function and gene expression at the desired endpoint. We achieve this convergence by utilizing efficient microfluidic trap arrays patterned with locally printed DNA barcodes, which allows us to both take images of single cells and prepare barcoded scRNA-seq libraries of the same cells. Building on the solid progress in our Phase I award, in which we demonstrated that the steps in our workflow are all technically feasible and compatible, which include the ability to print DNA barcodes inside sealed microfluidic chips, trap single cells in an array, acquire high-resolution images of each cell, and finally prepare cDNA libraries from barcoded primers attached to surfaces, in this Phase II, we will unify this workflow and demonstrate the ultimate goal of deploying our approach in a drug discovery platform that combines time lapse imaging and high-resolution transcriptome analysis of single cells. Our work plan is summarized in three specific aims. Our first aim is to show that thousands of uniquely barcoded PCR primers can be printed inside our chips in one unique primer per chamber format, and that these chips can be used in the preparation of high quality scRNA-seq libraries with low chamber cross-contamination. Our second aim is to demonstrate that this transcriptomics workflow can be successfully implemented after extended duration in vitro cell culture, during which time the barcodes are continually exposed to enzymes present in the serum and any extracellular secretions. Our third aim is to show that knowledge of the specific barcodes allows the transcripts derived from specific chambers to be selectively enriched during the pooled amplification steps – this will be used to demonstrate targeted sequencing of high-priority clones, which is of particular interest to our target customers. After all three aims have been individually demonstrated, they will be combined in a final demonstration showing the ability to identify drug-resistant clones and probe their transcriptomic signatures at high resolution. We expect this demonstration will excite potential customers and entice them to purchase Celldom instruments for drug development applications, therapeutics, and clinical diagnostics.