Nuclei isolation from adult mouse kidney for single-nucleus RNA-sequencing


  • J. Leiz
  • C. Hinze
  • A. Boltengagen
  • C. Braeuning
  • C. Kocks
  • N. Rajewsky
  • K.M. Schmidt-Ott


  • Journal of Visualized Experiments


  • J Vis Exp (175): e62901


  • 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.