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

摘要 持续激起学者和临床医生兴趣的反复出现的问题之一是理解 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， 神经性疾病、慢性感染和其他疾病。大规模并行的单细胞基因组工具可以 部分满足了这一市场需求；然而，科学界已明确表示，数字成绩单 统计不足，对提供更全面、多层次、多层次的工具有很强的拉动作用。 维单元格数据集。尤其需要高通量的单细胞表型工具， 由于使用活细胞的难度增加，它已经落后于基因组学工具的开发。至 为了满足这一需求，Celldom正在开发一种将基于图像的表型鉴定与单细胞鉴定相结合的平台 大规模转录转录是测量活细胞功能和基因表达所必需的 所需终结点。我们通过利用高效的微流控陷阱阵列来实现这种收敛，这些阵列在局部形成图案 打印的DNA条形码，使我们既可以拍摄单个细胞的图像，也可以准备条形码scRNA-seq 相同细胞的文库。在我们的第一阶段奖项取得坚实进展的基础上，我们证明了 我们工作流程中的所有步骤在技术上都是可行和兼容的，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，最后从附着在表面的条形码引物制备cdna文库，在这个阶段，我们将统一 并演示了在药物发现平台中部署我们的方法的最终目标 结合了时间推移成像和单细胞的高分辨率转录组分析。我们的工作计划是 总结为三个具体目标。我们的第一个目标是展示数千种独特的条码聚合酶链式反应 可以在我们的芯片内部以每室一种唯一的底漆格式打印，并且这些芯片可以用于 低小室交叉污染高质量scrna-seq文库的制备。我们的第二个目标是 证明这种转录工作流程可以在体外延长持续时间后成功实施 细胞培养，在此期间，条形码不断暴露在血清和任何 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允许记录 从特定的腔室中提取，在聚合扩增步骤中选择性地进行浓缩--这将是 用于演示高优先级克隆的定向排序，这是我们的目标特别感兴趣的 顾客。在所有三个目标单独演示后，它们将在决赛中合并 演示了识别耐药克隆并探测其转录签名的能力 高分辨率。我们预计这次演示将激发潜在客户的兴趣，并吸引他们购买 用于药物开发应用、治疗学和临床诊断学的CELLDOM仪器。

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.