Abstract: FR-PO940
Single Nucleus RNA-Seq from Fresh and Frozen Adult Mouse Kidney Offers Major Advantages over Single Cell RNA-Seq
Session Information
- Development, Stem Cells, Regenerative Medicine - II
October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Pathology and Lab Medicine
- 1501 Pathology and Lab Medicine: Basic
Authors
- Wu, Haojia, Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
- Kirita, Yuhei, Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
- Donnelly, Erinn L., Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
- Humphreys, Benjamin D., Renal Division Washington University School of Medicine, Saint Louis, Missouri, United States
Group or Team Name
- The Humphreys Lab
Background
Single-cell sequencing methods (scRNA-seq) have emerged as powerful tools for identification of cell types and states in kidney. However, generating a healthy single cell suspension is one of the biggest challenges to the field, limited by stress artifacts, RNA degradation and dissociation bias. We tested the hypothesis that single nucleus RNA-seq (snRNA-seq) is a superior approach.
Methods
We created 3,796 single cell transcriptomes from mouse kidney using the Dropseq platform, and single nucleus transcriptomes using the protocols from sNuc-Dropseq (2,951 nuclei), DroNc-seq (2,739 nuclei) and 10x Chromium (2,027 nuclei). We applied unbiased computational approaches to compare the gene expression and cell composition for each kidney cell type across different platforms. Finally, we aligned the cell types from snRNA-seq techniques to uncover the variations in gene expression within the shared subpopulations across techniques.
Results
12 clusters were identified in the scRNA-seq dataset with most clusters from tubule and no cells at all from glomerulus. One cluster specifically expressed genes that were previously defined as artifactual dissociation-induced stress response genes induced by the cell dissociation protocol. By contrast, snRNA-seq from all platforms captured a diversity of kidney cell types including glomerular podocytes, mesangial cells and endothelial cells. The artifact cell cluster was not observed in the snRNA-seq datasets since the procedure is carried out on ice. Integrated analysis revealed that all snRNA-seq techniques can detect the same kidney cell types but DroNc-seq can capture more transcripts at the same sequencing depth over other snRNA-seq techniques. We also demonstrate that snRNA-seq is feasible on snap frozen tissue.
Conclusion
snRNA-seq provides substantial advantages over scRNA-seq in kidney, including the absence of dissociation-induced transcriptional artifacts, better representation of glomerular cell types (reduced dissociation bias) and the ability to perform snRNA-seq on archival, frozen samples.
Funding
- NIDDK Support