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.

单细胞和单核测序技术是一个新兴领域，具有各种生物学、生物医学和临床应用。已经开发了许多高通量和低通量的方法来对单细胞的 RNA 和 DNA 含量进行测序。然而，对于所有这些方法，关键要求是单细胞或单核悬浮液的高质量输入。在处理易碎、存档的质量参差不齐的组织时，准备这样的悬浮液是一个限制步骤。这一障碍可能会阻止此类组织被单细胞技术广泛研究。我们描述了一种在几个小时内制备单核悬浮液的协议，该协议可靠地适用于使用标准实验室设备的多种死后和存档组织类型。该协议的阶段包括组织准备和解离、核提取以及核浓度评估和捕获。该协议可与其他已发布的协议相媲美，但不需要荧光辅助核分选 (FANS) 或超速离心。该协议可由熟悉基本实验室技术和设备的称职研究生执行。此外，这些制剂与使用 10X Genomics Chromium 系统的单核 (sn)RNA-seq 和转座酶可及染色质 (ATAC)-seq 分析兼容。该协议可靠地导致高效捕获高质量 snRNA-seq 库的单核。该协议可与其他已发布的协议相媲美，但不需要荧光辅助核分选 (FANS) 或超速离心。该协议可由熟悉基本实验室技术和设备的称职研究生执行。此外，这些制剂与使用 10X Genomics Chromium 系统的单核 (sn)RNA-seq 和转座酶可及染色质 (ATAC)-seq 分析兼容。该协议可靠地导致高效捕获高质量 snRNA-seq 库的单核。该协议可与其他已发布的协议相媲美，但不需要荧光辅助核分选 (FANS) 或超速离心。该协议可由熟悉基本实验室技术和设备的称职研究生执行。此外，这些制剂与使用 10X Genomics Chromium 系统的单核 (sn)RNA-seq 和转座酶可及染色质 (ATAC)-seq 分析兼容。该协议可靠地导致高效捕获高质量 snRNA-seq 库的单核。这些制剂与使用 10X Genomics Chromium 系统的单核 (sn)RNA-seq 和转座酶可及染色质 (ATAC)-seq 分析兼容。该协议可靠地导致高效捕获高质量 snRNA-seq 库的单核。这些制剂与使用 10X Genomics Chromium 系统的单核 (sn)RNA-seq 和转座酶可及染色质 (ATAC)-seq 分析兼容。该协议可靠地导致高效捕获高质量 snRNA-seq 库的单核。