(697c) Colony Formation within Hydrogel Microdroplets Enabled High Throughput Yeast Colony RNA-Seq

Yeasts have long been used as a model system for biological and biomedical research and played vital roles in biotechnology. While measuring genome-wide expression profile in a high throughput of yeasts is essential for both deepening our understanding of yeasts based biological and biomedical research and accelerating the characterization of yeasts in synthetic biology, such method has not been developed. This is because that bulk yeast culture RNA-seq is limited by the high cost and low throughput, and on the other hand, single yeast RNA-seq is technically challenging due to the low RNA content and difficulty of lysis. To circumvent these challenges, we developed a novel droplet microfluidic based high throughput single yeast colony RNA-seq workflow to enable the characterization of thousands of yeast colonies in parallel. Specifically, we first encapsulated single yeast cells into agarose hydrogel microdroplets using a flow focusing-splitter design followed by cultivating them into individual colonies. We then captured the RNA of individual colonies with barcoded RNA capture beads (Drop-seq beads) followed by reverse transcription, library preparation and next generation sequencing to collect single colony genome-wide expression profiles. The colony formation within hydrogel microdroplets amplifies the RNA content and enables effective yeast cell handling and lysis. The barcoded RNA capture with droplets microfluidic enables high throughput process and analysis. We first demonstrated the usage of this workflow by characterizing heterogeneity of genome-wide expression profile of over one thousand S. cerevisiae colonies. We further applied this workflow to analyze opaque-white switch of pathogenic yeast C. albican at single colony level. This method can be widely applied to enhance the understanding of yeasts based biological and biomedical research and also to accelerate the characterization of yeasts for synthetic biology.