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Well-paired-seq: a Size-exclusion and Locally Quasi-static Hydrodynamic Microwell Chip for Single-cell RNA-seq

ABSTRACT: Well-paired-seq: a Size-exclusion and Locally Quasi-static Hydrodynamic Microwell Chip for Single-cell RNA-seq

PROVIDER: PRJNA792809 | ENA |

REPOSITORIES: ENA

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Well-paired-seq: a Size-exclusion and Locally Quasi-static Hydrodynamic Microwell Chip for Single-cell RNA-seq

Project description:High throughput single-cell RNA sequencing (scRNA-seq) is a powerful technology for revealing new cell types and cellular states. However, the existing droplet or well-based methods are primarily based on the stochastic pairing of single cells and barcoded beads, resulting in a large number of empty microreactors and a low cell/bead utilization efficiency. Here, we present Well-paired-seq, consisting of thousands of size-exclusion and quasi-static hydrodynamic dual wells to address these limitations. The size-exclusion based dual-well structure allowed one cell and one bead to be trapped in the bottom well (cell-capture-well) and the top well (bead-capture-well), respectively, while the quasi-static hydrodynamic principle ensured that the trapped cells were unable to escape from cell-capture-wells, achieving cumulative capture of cells and buffer exchange in the Well-paired-seq chip. By the integration of quasi-static hydrodynamic and size-exclusion principles, the structure of the dual-well ensures one cell and one bead pairing with high density, acheiving high efficiency of cell/bead pairings (~80%) and low cell doublets. The high utilization of microreactors and single cells/beads enable us to achieve an ultra-high throughput (~105 cells ) with low collision rates (6.2%). We demonstrate the technical performance of Well-paired-seq by collecting transcriptome data from around 200,000 cells across 21 samples including various cell lines, peripheral blood mononuclear cells, and drug-treated cells, successfully revealing the heterogeneity of single cells and showing the wide applicability of Well-paired-seq for basic or clinical biomedical research.

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