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Protocol to obtain high-quality single-cell RNA-sequencing data from mouse liver cells using centrifugation

ABSTRACT: Summary High-quality single-cell RNA-sequencing data of liver cells, especially hepatocytes, are challenging due to cell death associated with hepatocyte isolation using fluorescence-activated cell sorting (FACS). Here, we present a protocol to obtain viable hepatocytes and nonparenchymal liver cells for scRNA-seq, using centrifugation. We detail steps for liver wash and enzyme perfusion, followed by in vitro dissociation of liver cells and gradient centrifugation. We further describe hepatocytes harvesting for subsequent viability check and scRNA-seq. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021) and Mederacke et al. (2015). Graphical abstract Highlights • Obtain high-quality single-cell sequencing data of hepatocytes• Concurrent isolation of non-parenchymal liver cells• Quick sample preparation without fluorescence-activated cell sorting• Application to mice fed with chow or high-fat high-sucrose diet Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. High-quality single-cell RNA-sequencing data of liver cells, especially hepatocytes, are challenging due to cell death associated with hepatocyte isolation using fluorescence-activated cell sorting (FACS). Here, we present a protocol to obtain viable hepatocytes and nonparenchymal liver cells for scRNA-seq, using centrifugation. We detail steps for liver wash and enzyme perfusion, followed by in vitro dissociation of liver cells and gradient centrifugation. We further describe hepatocyte cells harvesting for subsequent viability check and scRNA-seq.

SUBMITTER: Wang S

PROVIDER: S-EPMC9647703 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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