Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing

Marcus Alvarez; Jihane N Benhammou; Shuyun Rao; Lopa Mishra; Joseph R Pisegna; Päivi Pajukanta

doi:10.21769/BioProtoc.4601

Isolation of Nuclei from Human Snap-frozen Liver Tissue for Single-nucleus RNA Sequencing

Bio Protoc. 2023 Feb 5;13(3):e4601. doi: 10.21769/BioProtoc.4601.

Authors

Marcus Alvarez¹, Jihane N Benhammou^{2

3}, Shuyun Rao⁴, Lopa Mishra^{4

5

6}, Joseph R Pisegna^{1

3}, Päivi Pajukanta^{1

7

8}

Affiliations

¹ Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
² Vatche and Tamar Manoukian Division of Digestive Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
³ Division of Gastroenterology, Hepatology and Parenteral Nutrition, Department of Medicine, VA Greater Los Angeles Healthcare System, CA, USA.
⁴ Center for Translational Medicine, Department of Surgery, George Washington University, Washington DC, USA.
⁵ Institute for Bioelectronic Medicine, Feinstein Institutes for Medical Research; Divisions of Gastroenterology and Hepatology, Department of Medicine, Northwell Health, Manhasset, NY, USA.
⁶ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.
⁷ Bioinformatics Interdepartmental Program, UCLA, Los Angeles, CA, USA.
⁸ Institute for Precision Health, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

Abstract

Single-nucleus RNA sequencing (snRNA-seq) provides a powerful tool for studying cell type composition in heterogenous tissues. The liver is a vital organ composed of a diverse set of cell types; thus, single-cell technologies could greatly facilitate the deconvolution of liver tissue composition and various downstream omics analyses at the cell-type level. Applying single-cell technologies to fresh liver biopsies can, however, be very challenging, and snRNA-seq of snap-frozen liver biopsies requires some optimization given the high nucleic acid content of the solid liver tissue. Therefore, an optimized protocol for snRNA-seq specifically targeted for the use of frozen liver samples is needed to improve our understanding of human liver gene expression at the cell-type resolution. We present a protocol for performing nuclei isolation from snap-frozen liver tissues, as well as guidance on the application of snRNA-seq. We also provide guidance on optimizing the protocol to different tissue and sample types.

Keywords: Frozen tissue; Liver; Nuclei isolation; Single-nucleus RNA-seq.

Grants and funding

R01 DK132775/DK/NIDDK NIH HHS/United States