Shin David, Urbanek Madeleine E, Larson H Hanh, Moussa Anthony J, Lee Kevin Y, Baker Donovan L, Standen-Bloom Elio, Ramachandran Sangeetha, Bogdanoff Derek, Cadwell Cathryn R, Nowakowski Tomasz J
Biomedical Sciences Graduate Program, University of California, San Francisco, CA, USA.
Department of Neurological Surgery, University of California, San Francisco, CA, USA.
bioRxiv. 2024 Dec 11:2024.10.01.616167. doi: 10.1101/2024.10.01.616167.
Single cell genomics has revolutionized our understanding of neuronal cell types. However, scalable technologies for probing single-cell connectivity are lacking, and we are just beginning to understand how molecularly defined cell types are organized into functional circuits. Here, we describe a protocol to generate high-complexity barcoded rabies virus (RV) for scalable circuit mapping from tens of thousands of individual starter cells in parallel. In addition, we introduce a strategy for targeting RV-encoded barcode transcripts to the nucleus so that they can be read out using single-nucleus RNA sequencing (snRNA-seq). We apply this tool in organotypic slice cultures of the developing human cerebral cortex, which reveals the emergence of cell type-specific circuit motifs in midgestation. By leveraging the power and throughput of single cell genomics for mapping synaptic connectivity, we chart a path forward for scalable circuit mapping of molecularly-defined cell types in healthy and disease states.
单细胞基因组学彻底改变了我们对神经元细胞类型的理解。然而，用于探测单细胞连接性的可扩展技术尚不完善，而且我们才刚刚开始了解分子定义的细胞类型是如何组织成功能回路的。在这里，我们描述了一种生成高复杂性条形码狂犬病病毒（RV）的方案，用于从数万个单独的起始细胞中并行进行可扩展的回路映射。此外，我们还介绍了一种将RV编码的条形码转录本靶向细胞核的策略，以便可以使用单核RNA测序（snRNA-seq）对其进行读取。我们将此工具应用于发育中的人类大脑皮层的器官型切片培养中，这揭示了妊娠中期细胞类型特异性回路基序的出现。通过利用单细胞基因组学的能力和通量来绘制突触连接性，我们为在健康和疾病状态下对分子定义的细胞类型进行可扩展的回路映射绘制了一条前进的道路。
