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

使用液滴微流控以单细胞分辨率和高通量分析微生物细胞的转录异质性

• 批准号: 10002886
• 负责人: Anindita Basu
• 金额: $ 243万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-20 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10002886
• 关键词: Ablation; Acoustics; Antibiotic Resistance; Bacteria; Bar Codes; Biological; Cell Wall; Cells; Chemicals; Cytolysis; Development; Disease; Environment; Gene Expression Profiling; Genetic Transcription; Genomics; Heterogeneity; Hybrids; Individual; Inflammatory; Lasers; Libraries; Mammalian Cell; Measures; Medical; Messenger RNA; Methods; Microbe; Microfluidic Microchips; Microfluidics; Microscopy; Modernization; Molecular Biology; Population; Population Sizes; Process; Property; Proteomics; RNA; Reagent; Reporter Genes; Resolution; Sampling; Scheme; Scourge; Signal Transduction; Silicon; Tail; Techniques; Technology; Thick; Transcript; Translating; base; cost; cost effective; elastomeric; epigenomics; experimental study; fitness; microbial; microbial genomics; nanomaterials; novel; single-cell RNA sequencing; synthetic biology; targeted sequencing; tool; transcriptome sequencing

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.

项目摘要 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方法中可以看出。这种异质性可以是一种资产， 责任;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，抗生素耐药性的现代灾难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的分辨率。 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是不可行的。 不足以应对这一挑战。 最近的技术进步现在允许我们使用RNA-Seq来分析单个哺乳动物细胞。大规模和早期 条形码化之后再通过微流体汇集或操作，已经将规模增加到数万 信元然而，这些技术迄今未能转化为单一微生物细胞，原因如下： 单个微生物细胞裂解困难，特别是那些细胞壁较厚的细胞;（2）难以捕获和条形码化 相对稀疏的微生物mRNA，特别是当缺乏polyA尾（在细菌中）时;和（3）大群体 微生物种群的大小和复杂性需要在一个环境中采样数量级更多的细胞 实验 我们将利用液滴微流体技术，开发物理，化学和酶裂解方法， 研究新的分子生物学和测序技术，以开发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并对其进行条形码化。 微生物细胞;和（4）每个样品处理104-105个细胞，每个细胞具有数百个不同的转录物。 然后将条形码化的RNA合并并进行高深度测序。这些工具将是模块化的， 它的适用性超出了RNA-Seq，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，激光烧蚀，声波和等离子体共振。 拟议的项目将大大推进目前的技术，这些技术在吞吐量或 测量的RNA分子数量。目前，对于单个微生物细胞RNA还没有成功的策略- 大规模测序。我们的策略将使微生物中具有成本效益和通用的单细胞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体装置 将开发用于单个微生物细胞裂解、条形码化和文库制备的组件。