A Highly Sensitive, Accurate, and Automated Single-Cell RNA Sequencing Platform with Digital Microfluidics

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.