Profiling Transcriptional Heterogeneity in Microbial Cells at Single Cell resolution and High-throughput using Droplet Microfluidics

项目总结 已知微生物转录动力学在细胞之间是高度动态和异质的， 从基于显微镜和靶向测序的方法来看。这种异质性可以是一种资产，也可以是 从适应性的角度来看，转录异质性是在变化中生存的先决条件。 然而，现代抗生素耐药性的祸害可能归因于这种异质性。 在任何一种情况下，都有巨大的需求来表征转录动力学在分辨率 单个微生物细胞。然而，依赖于一个或几个选定的报告基因的传统方法 不足以应对这一挑战。 最近的技术进步现在使我们能够使用RNA-Seq来描述单个哺乳动物细胞。大规模和早期的 条形码之后的混合或微流控操作使规模增加到数万 细胞的数量。然而，到目前为止，由于(1)，这些技术未能转化为单一的微生物细胞 单个微生物细胞裂解困难，特别是细胞壁较厚的微生物；(2)难以捕获和条形码 相对稀疏的微生物mRNAs，特别是在缺乏Polya尾巴的情况下(在细菌中)；以及(3)大量种群 微生物种群的规模和复杂性，需要在一种 做实验。 我们将利用液滴微流体，开发物理、化学和酶促裂解方法，以及 研究新的分子生物学和测序技术以建立单细胞微生物基因组学 (1)分离和(2)裂解单个微生物细胞；(3)捕获单个微生物细胞的mRNA并对其进行条形码编码 微生物细胞；以及(4)每个样本处理104-105个细胞，每个细胞具有数百个不同的转录本。 条形码RNA随后将被汇集在一起，并在高深度进行测序。这些工具将是模块化的，并具有广泛的 超越RNA-Seq的适用性，包括单细胞表观基因组学和蛋白质组学。新的物理裂解模式 研究将包括MEMS、激光消融、声波和等离子体共振。 拟议中的项目将极大地推进当前的技术，这些技术在生产能力或 测量的RNA分子的数量。目前，还没有成功的单个微生物细胞RNA的策略。 规模上的SEQ。我们的策略将在微生物中实现经济高效和通用的单细胞RNA-Seq 巨大的吞吐量。含有硅、纳米材料和弹性体的新型混合微流控器件 将开发用于单个微生物细胞裂解、条形码和文库准备的组件。

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.