McGill Group for Suicide Studies, Douglas Mental Health University Institute, Montreal, Canada.
Department of Psychiatry, McGill University, Montreal, Canada.
Nat Protoc. 2021 Jun;16(6):2788-2801. doi: 10.1038/s41596-021-00514-4. Epub 2021 May 10.
Single-cell and single-nucleus sequencing techniques are a burgeoning field with various biological, biomedical and clinical applications. Numerous high- and low-throughput methods have been developed for sequencing the RNA and DNA content of single cells. However, for all these methods, the key requirement is high-quality input of a single-cell or single-nucleus suspension. Preparing such a suspension is the limiting step when working with fragile, archived tissues of variable quality. This hurdle can prevent such tissues from being extensively investigated with single-cell technologies. We describe a protocol for preparing single-nucleus suspensions within the span of a few hours that reliably works for multiple postmortem and archived tissue types using standard laboratory equipment. The stages of the protocol include tissue preparation and dissociation, nuclei extraction, and nuclei concentration assessment and capture. The protocol is comparable to other published protocols but does not require fluorescence-assisted nuclei sorting (FANS) or ultracentrifugation. The protocol can be carried out by a competent graduate student familiar with basic laboratory techniques and equipment. Moreover, these preparations are compatible with single-nucleus (sn)RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq using the 10X Genomics Chromium system. The protocol reliably results in efficient capture of single nuclei for high-quality snRNA-seq libraries.
单细胞和单细胞核测序技术是一个新兴领域，具有多种生物学、生物医学和临床应用。已经开发出许多高通量和低通量的方法来测序单细胞的 RNA 和 DNA 含量。然而，对于所有这些方法，关键要求是高质量的单细胞或单细胞核悬浮液输入。在处理脆弱的、质量不同的存档组织时，制备这种悬浮液是一个限制步骤。这一障碍可能会阻止这些组织广泛地使用单细胞技术进行研究。我们描述了一种在几个小时内制备单细胞核悬浮液的方案，该方案使用标准实验室设备可靠地适用于多种死后和存档组织类型。该方案包括组织准备和解离、细胞核提取以及细胞核浓度评估和捕获。该方案与其他已发表的方案相当，但不需要荧光辅助核分选（FANS）或超速离心。该方案可以由熟悉基本实验室技术和设备的有能力的研究生来执行。此外，这些制备物与使用 10X Genomics Chromium 系统的单细胞核 (sn)RNA-seq 和转座酶可及染色质 (ATAC)-seq 分析兼容。该方案可靠地实现了高效捕获高质量 snRNA-seq 文库的单细胞核。
