利用皮升级微室进行高效、高度并行的单细胞 mRNA 测序。

Highly parallel and efficient single cell mRNA sequencing with paired picoliter chambers.

机构信息

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.

Department of Chemistry, Stanford University, Stanford, CA, 94305, USA.

出版信息

Nat Commun. 2020 Apr 30;11(1):2118. doi: 10.1038/s41467-020-15765-0.

DOI:10.1038/s41467-020-15765-0

PMID:32355211

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7193604/

Abstract

ScRNA-seq has the ability to reveal accurate and precise cell types and states. Existing scRNA-seq platforms utilize bead-based technologies uniquely barcoding individual cells, facing practical challenges for precious samples with limited cell number. Here, we present a scRNA-seq platform, named Paired-seq, with high cells/beads utilization efficiency, cell-free RNAs removal capability, high gene detection ability and low cost. We utilize the differential flow resistance principle to achieve single cell/barcoded bead pairing with high cell utilization efficiency (95%). The integration of valves and pumps enables the complete removal of cell-free RNAs, efficient cell lysis and mRNA capture, achieving highest mRNA detection accuracy (R = 0.955) and comparable sensitivity. Lower reaction volume and higher mRNA capture and barcoding efficiency significantly reduce the cost of reagents and sequencing. The single-cell expression profile of mES and drug treated cells reveal cell heterogeneity, demonstrating the enormous potential of Paired-seq for cell biology, developmental biology and precision medicine.

摘要

单细胞 RNA 测序（scRNA-seq）能够揭示准确而精细的细胞类型和状态。现有的 scRNA-seq 平台利用基于珠粒的技术对单个细胞进行独特的条形码标记，但对于细胞数量有限的珍贵样本，这种方法面临着实际的挑战。在这里，我们提出了一种名为 Paired-seq 的 scRNA-seq 平台，它具有高细胞/珠粒利用率、无细胞 RNA 去除能力、高基因检测能力和低成本的特点。我们利用差分流动阻力原理实现了单细胞/条形码珠粒的高效配对，细胞利用率高达 95%。阀门和泵的集成实现了无细胞 RNA 的完全去除、高效的细胞裂解和 mRNA 捕获，从而实现了最高的 mRNA 检测准确性（R=0.955）和可比的灵敏度。较低的反应体积和更高的 mRNA 捕获和条形码效率显著降低了试剂和测序的成本。mES 细胞和药物处理细胞的单细胞表达谱揭示了细胞异质性，表明 Paired-seq 在细胞生物学、发育生物学和精准医学方面具有巨大的潜力。