High-throughput gene expression analysis with TempO-LINC sensitively resolves complex brain, lung and kidney heterogeneity at single-cell resolution

We report the development and performance of a novel genomics platform, TempO-LINC, for conducting high-throughput transcriptomic analysis on single cells and nuclei. TempO-LINC works by adding cell-identifying molecular barcodes onto highly selective and high-sensitivity gene expression probes within fixed cells, without having to first generate cDNA. Using an instrument-free combinatorial indexing approach, all probes within the same fixed cell receive an identical barcode, enabling the reconstruction of single-cell gene expression profiles across as few as several hundred cells and up to 100,000 + cells per sample. The TempO-LINC approach is easily scalable based on the number of barcodes and rounds of barcoding performed; however, for the experiments reported in this study, the assay utilized over 5.3 million unique barcodes. TempO-LINC offers a robust protocol for fixing and banking cells and displays high-sensitivity gene detection from multiple diverse sample types. We show that TempO-LINC has a multiplet rate of less than 1.1% and a cell capture rate of ~ 50%. Although the assay can accurately profile the whole transcriptome (19,683 human, 21,400 mouse and 21,119 rat genes), it can be targeted to measure only actionable/informative genes and molecular pathways of interest - thereby reducing sequencing requirements. In this study, we applied TempO-LINC to profile the transcriptomes of more than 90,000 cells across multiple species and sample types, including nuclei from mouse lung, kidney and brain tissues. The data demonstrated the ability to identify and annotate more than 50 unique cell populations and positively correlate expression of cell type-specific molecular markers within them. TempO-LINC is a robust new single-cell technology that is ideal for large-scale applications/studies with high data quality.