Neurons exhibit a rich diversity of morphological phenotypes, electrophysiological properties, and gene-expression patterns. Understanding how these different characteristics are interrelated at the single-cell level has been difficult because of the lack of techniques for multimodal profiling of individual cells. We recently developed Patch-seq, a technique that combines whole-cell patch-clamp recording, immunohistochemistry, and single-cell RNA-sequencing (scRNA-seq) to comprehensively profile single neurons from mouse brain slices. Here, we present a detailed step-by-step protocol, including modifications to the patching mechanics and recording procedure, reagents and recipes, procedures for immunohistochemistry, and other tips to assist researchers in obtaining high-quality morphological, electrophysiological, and transcriptomic data from single neurons. Successful implementation of Patch-seq allows researchers to explore the multidimensional phenotypic variability among neurons and to correlate gene expression with phenotype at the level of single cells. The entire procedure can be completed in ∼2 weeks through the combined efforts of a skilled electrophysiologist, molecular biologist, and biostatistician.

中文翻译：

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使用 Patch-seq 对单细胞形态、电生理学和基因表达进行多模式分析。

神经元表现出丰富的多样性的形态表型、电生理特性和基因表达模式。由于缺乏对单个细胞进行多模式分析的技术，了解这些不同特征在单细胞水平上如何相互关联一直很困难。我们最近开发了 Patch-seq，这是一种结合全细胞膜片钳记录、免疫组织化学和单细胞 RNA 测序 (scRNA-seq) 的技术，可全面分析小鼠脑切片中的单个神经元。在这里，我们提出了详细的分步方案，包括对修补机制和记录程序、试剂和配方、免疫组织化学程序的修改以及其他技巧，以帮助研究人员获得高质量的形态学、电生理学和转录组学数据单个神经元。Patch-seq 的成功实施使研究人员能够探索神经元之间的多维表型变异性，并在单细胞水平上将基因表达与表型相关联。通过熟练的电生理学家、分子生物学家和生物统计学家的共同努力，整个过程可以在约 2 周内完成。