Single-Bacterium Transcriptomics Using Nanoliter Microfluidic Droplets
EMSL Project ID
49046
Abstract
RNA-seq has been emerging as an advanced approach to the characterization and quantification of transcriptomes using next-generation sequencing technologies. Nevertheless, most studies target to large heterogeneous cell populations, and thus reflect the average transcriptomic behavior of all population components. Recently, RNA-seq from single eukaryotic cells has been established to discover global stochastic gene expression patterns, to analyze rare cells, as well as to identify novel cell types and functions. However, it is challenging to profile the transcriptomes of single prokaryotes (bacteria and archaea) owing to less RNA materials, short half-life time, the lack of poly(A) tails, and much smaller volume of prokaryotic cells. Here we propose to develop a nanoliter microfluidic droplet-based method to prepare cDNA samples from single bacterial cells for genome-wide RNA sequencing with high sensitivity, precision, accuracy and throughput. The ultra-low volume, high uniformity, and high controllability of microfluidic droplets can drastically enhance the efficiency and throughput of the reactions at the single-bacterium resolution without compromising measurement accuracy. In our method, the monodisperse agarose-in-oil droplets will be initially produced from a polydimethylsiloxane-glass microfluidic droplet generator manufactured with standard soft lithography. Meanwhile, statistically dilute single bacterial cells are compartmentalized into the droplets. Taking advantage of the unique thermo-responsive sol-gel capability of the agarose, we are able to transform the droplets into mechanically stable microgels which allow free exchange of small molecules between droplet interior and external solutions via diffusion, while encaging the high-molecular-weight components. Therefore, multiple reaction steps can be consecutively conducted in the droplets to lyse cells, release RNA, digest genomic DNA, enrich mRNA, and synthesize the first-strand cDNA. The cDNA is then amplified by massively parallel emulsion PCR in an oil-filled standard PCR tube using a conventional thermocycler, and the positive droplets are sorted using fluorescence-activated cell sorting. cDNA libraries are finally constructed and sequenced on SOLiD and Proton (Ion Torrent) sequencing platforms, followed by bioinformatics analysis. We will first validate this method with respect to sensitivity, precision/reproducibility and accuracy, and compare its performance with standard bulk methods and microliter single-cell methods using pure cell samples. Afterwards, the heterogeneous cell populations and contaminated cellular materials will be analyzed to test its selectivity and tolerance to environmental elements. The method is ultimately to be implemented to analyze samples collected from the natural environment such as soil. We envision that our method will provide a novel tool to comprehensive understanding of the diverse and ecology of complex microbial communities, as well as their functions and roles in terrestrial carbon cycling, climate change, plant drought resistance and productivity, and biofuel production.
Project Details
Start Date
2015-10-19
End Date
2018-09-30
Status
Closed
Released Data Link
Team
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