Summary/Abstract: Single cell gene expression assays have become critical tools for identifying functional subtypes of cells, as well as changes within specific subtypes resulting from development, disease or therapy treatment. However, most current methods are expensive, low in sample throughput and rely on oligo-dT primed reverse transcription. Furthermore, there is no method to-date that enables investigators to consistently measure low expressed genes from single cells, which prevents measurements of many key biomarkers and important genes and molecular pathways. Another problem with existing single-cell sequencing methods is that many of the cell-associated reads either fail to map to transcripts or map to uninformative ribosomal and mitochondrial genes. The aforementioned limitations have all contributed to most single-cell sequencing studies being restricted to relatively small numbers of samples and have prevented their wider scale adoption in biopharma, clinical, translational and applied markets. In Phase 1 SBIR development, we implemented a novel combinatorial split-pool-based workflow, TempO-LINC, that adds cell-identifying molecular barcodes onto high-sensitivity gene expression probes. All probes within the same cell receive an identical barcode, enabling the reconstruction of single-cell gene expression (and protein) profiles across tens of thousands of cells. TempO-LINC is an instrument free approach that multiplexes 96 1,000 cell samples per run, has a simple workflow that is amenable to automation and has the highest gene detection rate of any commercial single-cell platform. Critically, TempO-LINC reduces both the per sample assay cost as well as associated sequencing costs; furthermore, although we show the assay simultaneously profiles the whole transcriptome (23,000 transcripts), it can also be targeted to measure only genes/pathways of interest – dramatically reducing sequencing costs yet further. All of these features position TempO-LINC as a potential disruptive technology that researchers, clinicians and drug developers can use for new large-scale applications/studies using single-cell sequencing across thousands of samples while obtaining improved data quality. Our objective in this Phase 2 proposal is to take the basic TempO-LINC platform that we are currently running and ready it for commercialization by optimizing the sensitivity, workflow, multi-omic capability and software. To demonstrate the unique capabilities of TempO-LINC, we will benchmark it against 10X Chromium data using PBMCs and demonstrate a high-impact application of our technology with our collaborators/partners. Our approach relies on existing commercial TempO-Seq assays/technology, Phase 1 data that far exceeded all milestones, and a solid foundation of intellectual property. BioSpyder’s TempO-LINC constitutes a powerful single-cell platform with high potential for technical success, which will fill a critical need in the marketplace.