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

摘要/摘要：单细胞基因表达分析已成为鉴定功能的重要工具 细胞亚型，以及疾病或治疗引起的特定亚型内的变化。然而， 大多数当前的方法需要专用的硬件和套件，这使得它们非常昂贵，而且大多数方法是基于3英寸的 因此无法测量剪接变异体、基因融合、表达的单核苷酸变异体或非 多聚腺苷。此外，到目前为止，还没有一种方法能够使调查人员测量到最低甚至更多 来自单个细胞的适度表达的基因，这阻碍了许多关键生物标记物和 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 并不提供对这些基因丰度的定量测量，因此不能 用于测量使用批量制剂的表达水平沿可能的数量级的变化 它们也不能用于在单细胞水平上进行剂量反应实验。我们的方法将 使调查人员无需购买专有硬件即可进行单细胞分析，并提供 其性能可与块状细胞样品的分析相媲美。基于商业靶向基因 初步数据，将提供测量相同基因(低、中、高表达)和相似的数据 可以从大量样本中定量地测量基因/细胞As的数量，提供类似的动态 从大样本测量的计数的表达范围和正态分布。可以使用Tempo-LINC 一次处理几个100个单个细胞，最多100,000+个细胞。Tempo-LINC数据将作为基准 包括批量细胞数据和10倍基因组单细胞数据。此外，我们还将演示测量剪接的实用性 并提供单细胞剂量反应数据。这些数据将使用商用S1500生成 替代全转录组分析适应Tempo-LINC过程。在后续的第二阶段计划中， 将实施人和小鼠全转录组分析，Tempo-LINC分析商业化， 以及使用细胞系、纯化的细胞和从组织中分离的细胞展示的其他应用。这个 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 在单个细胞场内，超出了识别细胞亚群的范围。

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.