We propose to develop an intracellular stained Fluorescence Activated Cell Sorter Templated Oligonucleotide Sequencing whole transcriptome gene profiling assay, icsFACS/TempO-Seq, addressing an unmet need by enabling quantitative gene expression analysis of fixed, permeabilized, and intracellular stained and sorted cells (a method used to purify many different functionally important and rare subsets of cells). The assay will have the sensitivity to measure gene expression from small sets of cells and from single fixed and sorted cells whose function, fate or lineage are identified by cytosolic or nuclea biomarkers, including immune cells and circulating or tissue-dissociated stem and cancer cells. Having shown that TempO-Seq measurement is unimpaired by fixation, that antibody staining can be integrated into the TempO-Seq protocol, and that high quality data is obtained at the single cell level using RNA titrations, we propose to leverage a separately funded effort to implement a whole transcriptome TempO-Seq assay to develop and prove the feasibility of a whole transcriptome icsFACS/TempO-Seq assay. TempO-Seq effectively reduces the sequencing cost/sample to <$19/transcriptome (at third party vendor rates), compared to RNA-Seq which is typically several hundred dollars up to $2,200 (HiSeq) per transcriptome depending on the depth of sequencing. Investigators can measure a focused set of genes of fewer than 100 genes up to the whole transcriptome, plus measure specific mutations or gene fusions with icsFACS/TempO-Seq. We will implement the assay, determine its performance (sensitivity, reproducibility, dynamic range), and then perform gene expression profiling of human T cell subsets using anti-CD3/28 activated human peripheral blood lymphocytes which are stained for a combination of surface (CD3,4,8,25) and intracellular (TBX21, eomesodermin, Foxp3) antigens, and then FACS-sorted. Analysis of single cell preps and small homogeneous populations will generate important novel data, with single cell gene expression being used to define additional molecular phenotypes, reveal molecular pathways, and provide insight in the overall regulation of single cell transcription. Based on this feasibility the commercial development, validation, and launch of icsFACS/TempO-Seq will be carried out in Phase II. Flow cytometry and FACS sorting remains a robust and growing market into which BioSpyder will sell icsFACS/TempO-Seq kits. No specialized hardware is required to perform the assay, nor is extensive bioinformatics required for data analysis, making this assay easy to adopt.
We propose to develop and demonstrate the feasibility and utility of an intracellular stained Fluorescence Activated Cell Sorter (icsFACS) Templated Oligonucleotide Sequencing (icsFACS/TempO-Seq) whole transcriptome gene expression profiling assay, to monitor gene expression from fixed, permeabilized, and intracellularly stained and sorted cells. icsFACS/TempO-Seq will address an unmet need within the large and growing flow cytometry market to profile expression of focused sets of genes up to the whole transcriptome in a cost effective and easy to adopt manner from subpopulations and single cells that can only be identified using intracellular staining of biomarkers, such as cells critical in te immune response pathway, cancer and other diseases. We will demonstrate the feasibility of profiling gene expression from single cells and small populations to define molecular phenotypes and pathways of activation and differentiation using anti CD3/CD28 activated human T cell subsets defined using intracellular antibody staining of the transcription factors TBX21, eomesodermin, and Foxp3, respectively, and surface staining for a combination of markers (CD3,4,8,25), generating novel data regarding the molecular phenotypes and mechanisms of T cell subsets that can only be identified using intracellular staining, and of individual cells within those subsets.
