RNA cytometry of single-cells using semi-permeable microcapsules.

Analytical tools for gene expression profiling of individual cells are critical for studying complex biological systems. However, the techniques enabling rapid measurements of gene expression on thousands of single-cells are lacking. Here, we report a high-throughput RNA cytometry for digital profiling of single-cells isolated in liquid droplets enveloped by a thin semi-permeable membrane (microcapsules). Due to the selective permeability of the membrane, the desirable enzymes and reagents can be loaded, or replaced, in the microcapsule at any given step by simply changing the reaction buffer in which the microcapsules are dispersed. Therefore, complex molecular biology workflows can be readily adapted to conduct nucleic acid analysis on encapsulated mammalian cells, or other biological species. The microcapsules support sequential multi-step enzymatic reactions and remain intact under different biochemical conditions, freezing, thawing, and thermocycling. Combining microcapsules with conventional FACS provides a high-throughput approach for conducting RNA cytometry of individual cells based on their digital gene expression signature.

Multi-step processing of single cells using semi-permeable capsules.

Droplet microfluidics technology provides a powerful approach to isolate and process millions of single cells simultaneously. Despite many exciting applications that have emerged based on this technology, workflows based on multi-step operations, including molecular biology and cell-based phenotypic screening assays, cannot be easily adapted to droplet format. Here, we present a microfluidics-based technique to isolate single cells, or biological samples, into semi-permeable hydrogel capsules and perform multi-step biological workflows on thousands to millions of individual cells simultaneously. The biochemical reactions are performed by changing the aqueous buffer surrounding the capsules, without needing sophisticated equipment. The semi-permeable nature of the capsules' shell retains large encapsulated biomolecules (such as genome) while allowing smaller molecules (such as proteins) to passively diffuse. In contrast to conventional hydrogel bead assays, the approach presented here improves bacterial cell retention during multi-step procedures as well as the efficiency of biochemical reactions. We showcase two examples of capsule use for single genome amplification of bacteria, and expansion of individual clones into isogenic microcolonies for later screening for biodegradable plastic production.