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

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.Project Narrative Singe cell gene expression assays have become important tools to identify functional subtypes of cells and changes in the numbers of cells within each subtype, or of subtypes, resulting from disease and treatments. However, current methods require dedicated hardware and kits, making them expensive, and do not permit low expressed genes that may be key biomarkers to be measured, typically only measuring a limited number of genes/cell, typically not providing quantitative measurements of abundance or changes in expression level, and are often 3’ based and so cannot measure splice variants or RNA such as long non-coding RNA that are not polyadenylated. We will demonstrate the feasibility of a novel TempO-LINC assay using a barcoding approach that does not require proprietary hardware, and demonstrate that this assay provides single cell data that measures the low and moderately expressed genes identified from bulk samples, is highly correlated to bulk cell benchmark assay data with respect to the number of genes/cell, represents the dynamic expression range of those genes within each cell, and can provide single cell dose response data - enabling single cell studies not previously possible to understand (e.g.) molecular mechanistic differences at the single cell level of disease, and drug efficacy and safety.