Yin Kun, Zhao Meijuan, Lin Li, Chen Yingwen, Huang Shanqing, Zhu Chun, Liang Xuan, Lin Fanghe, Wei Haopai, Zeng Huimin, Zhu Zhi, Song Jia, Yang Chaoyong
State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Key Laboratory for Chemical Biology of Fujian Province, Collaborative Innovation Center of Chemistry for Energy Materials, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, P. R. China.
Institute of Molecular Medicine, State Key Laboratory of Oncogenes and Related Genes, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200120, China.
Small Methods. 2022 Jul;6(7):e2200341. doi: 10.1002/smtd.202200341. Epub 2022 May 6.
Single-cell RNA sequencing (scRNA-seq) is a powerful technology for revealing the heterogeneity of cellular states. However, existing scRNA-seq platforms that utilize bead-based technologies suffer from a large number of empty microreactors and a low cell/bead capture efficiency. Here, Well-paired-seq is presented, which consists of thousands of size exclusion and quasi-static hydrodynamic dual wells to address these limitations. The size-exclusion principle allows 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 ensures that the trapped cells are difficult to escape from cell-capture-wells, achieving cumulative capture of cells and effective buffer exchange. By the integration of quasi-static hydrodynamic and size-exclusion principles, the dual wells ensure single cells/beads pairing with high density, achieving excellent efficiency of cell capture (≈91%), cell/bead pairing (≈82%), and cell-free RNA removal. The high utilization of microreactors and single cells/beads enable to achieve a high throughput (≈10 cells) with low collision rates. The technical performance of Well-paired-seq is demonstrated by collecting transcriptome data from around 200 000 cells across 21 samples, successfully revealing the heterogeneity of single cells and showing the wide applicability of Well-paired-seq for basic and clinical research.
单细胞RNA测序（scRNA-seq）是一种揭示细胞状态异质性的强大技术。然而，现有的利用基于微珠技术的scRNA-seq平台存在大量空的微反应器以及细胞/微珠捕获效率低的问题。在此，介绍了Well-paired-seq，它由数千个尺寸排阻和准静态流体动力学双阱组成，以解决这些限制。尺寸排阻原理允许一个细胞和一个微珠分别被困在底部阱（细胞捕获阱）和顶部阱（微珠捕获阱）中，而准静态流体动力学原理确保被困细胞难以从细胞捕获阱中逸出，实现细胞的累积捕获和有效的缓冲液交换。通过整合准静态流体动力学和尺寸排阻原理，双阱确保单细胞/微珠以高密度配对，实现优异的细胞捕获效率（约91%）、细胞/微珠配对效率（约82%）和无细胞RNA去除效率。微反应器和单细胞/微珠的高利用率使得能够以低碰撞率实现高通量（约10个细胞）。通过从21个样本中收集约200000个细胞的转录组数据，展示了Well-paired-seq的技术性能，成功揭示了单细胞的异质性，并表明Well-paired-seq在基础和临床研究中具有广泛的适用性。
