Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Centre Basel.
Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Centre Basel;
J Vis Exp. 2023 Jul 28(197). doi: 10.3791/65611.
Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.
单细胞和单细胞核 RNA 测序由于提供了丰富的转录组信息，已成为常见的实验室应用。特别是单细胞核 RNA 测序，对于研究难以分离组织中的基因表达非常有用。此外，这种方法也与冷冻（存档）材料兼容。在这里，我们描述了一种从冷冻哺乳动物组织中分离高质量单细胞核的方案，该方案使用商业上可获得的仪器和试剂以部分自动化的方式进行下游单细胞核 RNA 测序。具体来说，使用机器人匀浆器来自动化和标准化组织匀浆，然后使用优化的化学梯度过滤细胞核。最后，我们使用自动化荧光细胞计数器准确和自动地计数细胞核。该方案在小鼠脑、大鼠肾和食蟹猴肝脾组织上进行了验证。该方案简单、快速，并且无需广泛优化即可轻松适用于各种哺乳动物组织，为下游单细胞核 RNA 测序提供了高质量的细胞核。
