A Highly Sensitive, Accurate, and Automated Single-Cell RNA Sequencing Platform with Digital Microfluidics
Xing Xu, Qianqian Zhang, Jia Song, Qingyu Ruan, Weidong Ruan, Yujie Chen, Jian Yang, Xuebing Zhang, Yanling Song, Zhi Zhu, Chaoyong Yang
Abstract
Single-cell RNA sequencing (scRNA-seq) is a powerful method in investigating single-cell heterogeneity to reveal rare cells, identify cell subpopulations, and construct a cell atlas. Conventional benchtop methods for scRNA-seq, including multistep operations, are labor intensive, reaction inefficient, contamination prone, and reagent consuming. Here we report a digital microfluidics-based single-cell RNA sequencing (digital-RNA-seq) for simple, efficient, and low-cost single-cell mRNA measurements. Digital-RNA-seq automates fluid handling as discrete droplets to sequentially perform protocols of scRNA-seq. To overcome the current problems of single-cell isolation in efficiency, integrity, selectivity, and flexibility, we propose a new strategy, passive dispensing method, relying on well-designed hydrophilic-hydrophobic microfeatures to rapidly generate single-cell subdroplets when a droplet of cell suspension is encountered. For sufficient cDNA generation and amplification, digital-RNA-seq uses nanoliter reaction volumes and hydrophobic reaction interfaces, achieving high sensitivity in gene detection. Additionally, the stable droplet handling and oil-closed reaction space featured in digital-RNA-seq ensure highly accurate measurement. We demonstrate the functionality of digital-RNA-seq by quantifying heterogeneity among single cells, where digital-RNA-seq shows excellent performance in rare transcript detection, cell type differentiation, and essential gene identification. With the advantages of automation, sensitivity, and accuracy, digital-RNA-seq represents a promising scRNA-seq platform for a wide variety of biological applications.