A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis.
A robust method to isolate Drosophila fat body nuclei for transcriptomic analysis.
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DOI：
10.1080/19336934.2021.1978776
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发表时间：
2022-12
期刊：
影响因子：
1.2
通讯作者：
Lazzaro BP
中科院分区：
文献类型：
--
作者：
Gupta V;Lazzaro BP
基因表达谱通常在组织水平上描述，或者在果蝇中，通常在整个生物体的水平上描述。将整个组织的基因表达分解为单个测量，平均了构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学评估。然而，sc-RNAseq需要高质量的活细胞或细胞核悬浮液，而许多重要组织仍然缺乏产生健康细胞和细胞核的细胞分离方法。昆虫脂肪体是一个多功能组织，负责多种生理过程，因此是sc-RNAseq的重要靶点。果蝇成年脂肪体由脆弱的细胞组成，这些细胞在维持细胞活力的同时难以分离。作为一种替代方法，我们开发了一种分离单个脂肪体核用于RNA-seq的方法。我们的分离方法在很大程度上没有线粒体污染，与其他细胞核制备方法相比，每个细胞核的转录本捕获率更高。我们的方法可以很好地用于单细胞细胞核测序，并且可以潜在地用于批量rna测序。
Gene expression profiles are typically described at the level of the tissue or, often in Drosophila, 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 Drosophila 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.
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