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| Item Type: | Article |
|---|---|
| Title: | Nuclei isolation from adult mouse kidney for single-nucleus RNA-sequencing |
| Creators Name: | Leiz, J. and Hinze, C. and Boltengagen, A. and Braeuning, C. and Kocks, C. and Rajewsky, N. and Schmidt-Ott, K.M. |
| Abstract: | The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla. Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 (°)C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research. |
| Keywords: | Cell Nucleus, Kidney, RNA, RNA Sequence Analysis, Transcriptome, Animals, Mice |
| Source: | Journal of Visualized Experiments |
| ISSN: | 1940-087X |
| Publisher: | JoVE |
| Number: | 175 |
| Page Range: | e62901 |
| Date: | September 2021 |
| Official Publication: | https://doi.org/10.3791/62901 |
| PubMed: | View item in PubMed |
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