Nguyen Tho Huu, Vicidomini Rosario, Choudhury Saumitra Dey, Coon Steven L, Iben James, Brody Thomas, Serpe Mihaela
Section on Cellular Communication, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland.
Molecular Genomics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institutes of Health (NIH), Bethesda, Maryland.
Curr Protoc. 2021 Feb;1(2):e38. doi: 10.1002/cpz1.38.
Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single-cell suspensions Basic Protocol 2: Preparation and sequencing of single-cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.
果蝇提供了一个强大的遗传系统和一个研究神经系统发育与功能的绝佳模型。果蝇的小大脑和复杂行为对于绘制神经回路以及阐明行为的神经基础起到了重要作用。果蝇发育的快速进程以及丰富的遗传工具使得对细胞分化和命运决定的系统性研究成为可能，并且揭示了轴突导向和靶向的策略。神经元结构的可及性以及在选择性细胞和/或突触区室中编辑和操纵基因表达的能力揭示了突触组装和神经元连接的机制。单细胞RNA测序（scRNA-seq）的最新进展进一步增强了我们对果蝇大脑中神经元多样性的认识和理解。然而，由于果蝇大脑及其组成细胞的体积较小，scRNA-seq方法需要进行一些调整。在这里，我们描述了一套针对果蝇幼虫腹神经索scRNA-seq分析优化的方案，从组织解剖和细胞解离到cDNA文库制备、测序和数据分析。我们将此工作流程应用于三个独立的样本，并详细说明了将scRNA-seq成功应用于神经元多样性研究相关的技术挑战。一篇配套文章（Vicidomini、Nguyen、Choudhury、Brody和Serpe，2021年）提出了一个定制的多阶段分析流程，该流程整合了不同R包中包含的模块，以确保进行高灵活性、高质量的RNA-seq数据分析。这些方案是针对果蝇幼虫腹神经索开发的，但可以很容易地适用于其他组织和模式生物。©2021美国政府。基本方案1：幼虫腹神经索的解剖和单细胞悬液的制备 基本方案2：单细胞转录组文库的制备和测序 基本方案3：将原始测序数据比对到索引基因组并生成计数矩阵。
