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Protocol to obtain high-quality single-cell RNA-sequencing data from mouse liver cells using centrifugation
1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
STAR Protocols
|November 17, 2022
Summary
This study introduces a new protocol for isolating viable liver cells, including hepatocytes, for single-cell RNA sequencing (scRNA-seq). This method avoids cell death often seen with fluorescence-activated cell sorting (FACS).
Area of Science:
- Hepatology
- Molecular Biology
- Genomics
Background:
- High-quality single-cell RNA sequencing (scRNA-seq) of liver cells is crucial for understanding liver biology.
- Hepatocyte isolation for scRNA-seq is often hampered by cell death, particularly when using fluorescence-activated cell sorting (FACS).
Purpose of the Study:
- To develop and present a protocol for obtaining viable hepatocytes and nonparenchymal liver cells suitable for scRNA-seq.
- To overcome the limitations of existing cell isolation methods that lead to significant cell death.
Main Methods:
- The protocol involves liver wash and enzyme perfusion.
- It includes in vitro dissociation of liver cells and gradient centrifugation.
- Hepatocytes are harvested for subsequent viability checks and scRNA-seq.
Main Results:
- The presented protocol yields viable hepatocytes and nonparenchymal liver cells.
- This method is an alternative to FACS for obtaining high-quality scRNA-seq data from liver cells.
Conclusions:
- This protocol provides a reliable method for preparing viable liver cells for scRNA-seq.
- It enables comprehensive analysis of liver cell populations without compromising cell integrity.
