PROJECT SUMMARY Microbial transcriptional dynamics have been known to be highly dynamic and heterogeneous between cells, seen from microscopy based and targeted sequencing approaches. Such heterogeneity can be an asset or a liability; from a fitness perspective, transcriptional heterogeneity is a prerequisite for survival under changing environments; however, the modern scourge of antibiotic resistance may be ascribed to such heterogeneities. In either case, there is an enormous need to characterize the transcriptional dynamics at the resolution of individual microbial cell. However traditional approaches relying on one or a few selected reporter genes are inadequate for this challenge. Recent technical advances now allow us to use RNA-Seq to profile single mammalian cells. Massive and early barcoding followed by pooling or manipulation by microfluidics have increased the scale to tens of thousands of cells. However, these technologies have thus far failed to translate to single microbial cells due to (1) difficulty in single microbial cell lysis, especially those with thick cell wall; (2) difficulty to capture and barcode relatively sparse microbial mRNAs, especially when lacking polyA tails (in bacteria); and (3) large population size and complexity of microbial population that require orders of magnitude more cells be sampled in an experiment. We will leverage droplet microfluidics, develop physical, chemical and enzymatic lysis methods, and investigate novel molecular biology and sequencing techniques to develop a single-cell microbial genomics pipeline to (1) Isolate and (2) lyse single microbial cells; (3) capture and barcode the mRNA of single microbial cells; and (4) process 104-105 cells per sample with hundreds of distinct transcripts per cell. Barcoded RNA will then be pooled and sequenced at high depth. These tools will be modular and have broad applicability beyond RNA-Seq, including single cell epigenomics and proteomics. New physical lysis modes investigated will include MEMS, laser ablation, acoustic waves, and plasmon resonance. The proposed project will significantly advance current technologies which are limited in throughput or the number of RNA molecules measured. Currently, there is no successful strategy for single microbial cell RNA- Seq at scale. Our strategy will enable cost-effective and generalized single-cell RNA-Seq in microbes at massive throughput. Novel, hybrid microfluidic devices containing silicon, nanomaterials and elastomeric components will be developed for single microbial cell lysis, barcoding and library prep.

PROJECT NARRATIVE Microbial transcription has been shown to be highly dynamic and heterogeneous between cells, using targeted approaches like microscopy and microarrays; this heterogeneity and dynamics have serious medical implications in antibiotic resistance and inflammatory disorders as well as commercial relevance in synthetic biology. Progress in single microbial cell genomics has been hindered due to challenges in 1) sheer variety of microbial taxa, species and strain-specific biological properties, 2) lysis of tough cell walls, and 3) low signal from relatively sparse microbial mRNA. I propose to combine recent developments in single cell genomics with new lysis and sequencing schemes to develop a generalized single-cell microbial RNA-seq pipeline that will allow us to process ~104-105 cells and capture ~500 transcripts per cell at reduced reagent and sequencing costs.