Laboratory of Molecular and Behavioral Neuroscience, Institute for Neuroscience, Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland.
Neuroscience Center Zurich, ETH Zurich and University of Zurich, Zurich, Switzerland.
Curr Protoc. 2023 Nov;3(11):e919. doi: 10.1002/cpz1.919.
Neuroscience research greatly benefits from single-cell sequencing technologies, which can reveal transcriptional alterations on a cellular level. However, preparing single-cell suspensions is technically challenging, requires experience, and has several limitations that can influence the transcriptional readout. Performing sequencing of single nuclei instead of single cells alleviates several of the challenges of sample preparation and highlights acute nuclear transcription. Here, we provide a protocol to prepare a nuclei suspension for single-nucleus RNA-sequencing for cell type-specific transcriptional profiling of brain tissue using the 10x Genomics single-cell gene expression assay. Furthermore, we highlight important aspects to consider during experimental design and data analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of single-nucleus suspension Basic Protocol 2: Preparation and sequencing of single-nucleus libraries for RNA-seq.
神经科学研究极大地受益于单细胞测序技术，该技术可以揭示细胞水平上的转录变化。然而，制备单细胞悬液在技术上具有挑战性，需要经验，并且存在一些可能影响转录读数的限制。替代单细胞进行单个核测序可以减轻样品制备的一些挑战，并突出急性核转录。在这里，我们提供了一种使用 10x Genomics 单细胞基因表达分析试剂盒为单细胞 RNA 测序制备核悬浮液的方案，用于对脑组织进行特定细胞类型的转录谱分析。此外，我们还强调了在实验设计和数据分析过程中需要考虑的重要方面。© 2023 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：制备单细胞悬液基本方案 2：制备和测序用于 RNA-seq 的单个核文库。
