TempO-LINC high throughput, high sensitivity single cell gene expression profiling assay

摘要：单细胞基因表达检测已经成为鉴定功能性的重要工具。 细胞的亚型，以及由疾病或治疗引起的特定亚型内的变化。但是，在这方面， 大多数当前的方法需要专用的硬件和工具包，这使得它们昂贵，并且大多数是基于3'的， 因此，不能测量剪接变体、基因融合、表达的单核苷酸变体或不表达的RNA。 聚腺苷酸化。此外，目前还没有一种方法能够使调查人员测量低甚至多个 来自单细胞的中度表达基因，这阻止了许多关键生物标志物的测量， 在分子途径和功能中的重要基因。目前的方法也只能测量有限数量的 基因/细胞，并且不提供那些基因的丰度的定量测量，因此不能被 用于测量表达水平沿着可能的数量级的变化， 细胞，也不能用于进行单细胞水平的剂量反应实验。我们的方法将 使研究者能够在不购买专有硬件的情况下进行单细胞测定， 的性能可与散装电池样品的剖析相媲美。基于商业靶向基因 表达TempO-Seq®批量细胞测定，我们将实施TempO-LINC单细胞测定，其基于 初步数据，将提供测量相同基因（低，中，高表达）和类似基因的数据。 可以从大量样品定量测量的基因/细胞的数量，提供类似的动态 表达范围和正态分布的计数，从散装样品测量。可以使用TempO-LINC 一次处理100个单细胞到100，000+细胞。TempO-LINC数据将根据 大细胞数据和10 x Genomics单细胞数据。此外，我们还将演示测量剪接的实用性 变体并提供单细胞剂量反应数据。这些数据将使用商用S1500生成 适用于TempO-LINC过程的替代全转录组测定。在后续的第二阶段计划中， 将实施人和小鼠全转录组测定，将TempO-LINC测定商业化， 以及使用细胞系、纯化细胞和从组织解离的细胞证明的其它应用。的 TempO-LINC单细胞分析的性能和能力将推动所追求的应用的扩展 在单细胞领域内，除了细胞亚群的鉴定之外。

Summary/Abstract: Single cell gene expression assays have become important tools to identify functional subtypes of cells, as well as changes within specific subtypes resulting from diseases or treatments. However, most current methods require dedicated hardware and kits, making them expensive, and most are 3’ based and thus cannot measure splice variants, gene fusions, expressed single nucleotide variants, or RNAs that are not polyadenylated. Furthermore, there is no method to-date that enables investigators to measure low or even many moderately expressed genes from single cells, which prevents measurements of many key biomarkers and important genes in molecular pathways and functions. Current methods also only measure a limited number of genes/cell and do not provide quantitative measurements of the abundance of those genes, and thus cannot be used to measure changes in expression level along the order of magnitude possible using bulk preparations of cells, nor can they be used to carry out dose response experiments at the single cell level. Our approach will enable investigators to carry out single cell assays without purchase of proprietary hardware, and provide a level of performance comparable to the profiling of bulk cell samples. Based on the commercial targeted gene expression TempO-Seq® bulk cell assay, we will implement a TempO-LINC single cell assay which, based on preliminary data, will provide data measuring the same genes (low, moderate, high expressed) and similar number of genes/cell as can be measured from bulk samples, quantitatively, providing a similar dynamic expression range and normal distribution of counts as measured from bulk samples. TempO-LINC can be used to process a few 100 single cells up to 100,000+ cells at a time. TempO-LINC data will be benchmarked against both bulk cell data and 10x Genomics single cell data. Furthermore, we will demonstrate utility to measure splice variants and to provide single cell dose response data. These data will be generated using the commercial S1500 surrogate whole transcriptome assay adapted to the TempO-LINC process. In a follow-on Phase II program, the human and mouse whole transcriptome assays will be implemented, the TempO-LINC assay commercialized, and additional applications demonstrated using cell lines, purified cells, and cells dissociated from tissues. The performance and capabilities of the TempO-LINC single cell assay will drive expansion of applications pursued within the single cell field beyond identification of subpopulations of cells.