Department of Entomology, Cornell University, Ithaca, NY, USA.
Cornell Institute of Host-Microbe Interactions and Disease, Cornell University, Ithaca, NY, USA.
Fly (Austin). 2022 Dec;16(1):62-67. doi: 10.1080/19336934.2021.1978776.
Gene expression profiles are typically described at the level of the tissue or, often in , at the level of the whole organism. Collapsing the gene expression of entire tissues into single measures averages over potentially important heterogeneity among the cells that make up that tissue. The advent of single-cell RNA-sequencing technology (sc-RNAseq) allows transcriptomic evaluation of the individual cells that make up a tissue. However, sc-RNAseq requires a high-quality suspension of viable cells or nuclei, and cell dissociation methods that yield healthy cells and nuclei are still lacking for many important tissues. The insect fat body is a polyfunctional tissue responsible for diverse physiological processes and therefore is an important target for sc-RNAseq. The adult fat body consists of fragile cells that are difficult to dissociate while maintaining cell viability. As an alternative, we developed a method to isolate single fat body nuclei for RNA-seq. Our isolation method is largely free of mitochondrial contamination and yields higher capture of transcripts per nucleus compared to other nuclei preparation methods. Our method works well for single-cell nuclei sequencing and can potentially be implemented for bulk RNA-seq.
基因表达谱通常在组织水平上进行描述，或者通常在整个生物体的水平上进行描述。将整个组织的基因表达压缩成单个指标，会平均掩盖组成该组织的细胞之间潜在的重要异质性。单细胞 RNA 测序技术（sc-RNAseq）的出现允许对组成组织的单个细胞进行转录组评估。然而，sc-RNAseq 需要高质量的、可存活细胞或核的悬浮液，并且对于许多重要的组织，仍然缺乏产生健康细胞和核的细胞解离方法。昆虫脂肪体是一种多功能组织，负责多种生理过程，因此是 sc-RNAseq 的重要目标。成年脂肪体由易碎的细胞组成，在保持细胞活力的同时很难分离。作为替代方法，我们开发了一种用于 RNA-seq 的分离单个脂肪体核的方法。我们的分离方法基本上没有线粒体污染，与其他核制备方法相比，每个核的转录本捕获量更高。我们的方法适用于单细胞核测序，并且有可能适用于批量 RNA-seq。
