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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). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.