Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.
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.
Kidneys display a highly complex tissue architecture. They consist of functionally and anatomically distinct segments along a corticomedullary axis and mediate biological functions, such as regulation of extracellular fluid volume, electrolyte balance, or acid-base homeostasis1.
Advances in single-cell transcriptomics have enabled the in-depth characterization of complex tissues and accelerated the understanding of segment and cell type-specific gene expression in renal physiology, development, and disease2,3,4.
However, the enzyme-based dissociation protocols that are frequently utilized for scRNA-seq display several drawbacks and constraints. Depending on the protocol, they generate transcriptional stress responses and tissue dissociation bias towards easier-to-dissociate cortical cell types5,6. Although protocols using cold-active proteases for embryonic kidneys are able to mitigate stress-related transcriptional alterations, they fail to overcome the dissociation bias towards cortical cells and might not be readily adaptable to different kinds of diseased kidney tissues7. In addition, single-cell approaches are not easily compatible with frozen tissue samples, limiting their application mostly to non-archived, fresh tissue, thus making the tissue collection a restricting factor6.
Single-nuclei RNA sequencing (snRNA-seq) can circumvent these limitations8,9. Here, we present a protocol for nuclei isolation from a central slice of frozen adult mouse kidney tissue (Figure 1)10. Our protocol is simple and provides a standardized approach to obtain RNA sequencing libraries with a balanced representation of diverse kidney cell types for experimental models that do not involve strong regional tissue changes. In the latter case, our protocol can also be performed with whole kidneys.
Single-cell transcriptomics advance the understanding of cell type-specific gene expression in renal physiology and disease. Here, we provided a simple and reproducible method to isolate high-quality single nuclei from frozen mouse kidney tissue for snRNA-seq in a standardized way.
For snRNA-seq, it is critical to use high-quality nuclei as input for library generation and to avoid RNA degradation during tissue processing. Therefore, the incubation of tissue pieces in RNA stabilization solution immediately after dissection is essential to protect and stabilize cellular RNA and allows to store samples at – 80 °C indefinitely. When applying this protocol to frozen tissue without RNA stabilization solution treatment, such as archival material, a trial run is required, and the RNA quality needs to be assessed as we observed a significant loss of RNA integrity in snap-frozen tissue without prior incubation in RNA stabilization solution.
In general, appropriate sample handling is crucial to maximizing the recovery of intact, single nuclei. All resuspension steps should be carried out by pipetting carefully to avoid shear stress and physical damage. Buffers for the final nuclei resuspension and nuclei sorting should contain BSA to avoid nuclei loss and aggregation.
Buffer volumes in this protocol are optimized for very small tissue samples (~15 mg). It is critical to ensure complete cell lysis and sufficient washing to generate high-quality suspensions. Larger tissue blocks or whole kidney samples will result in excessive nuclei concentrations that lead to clumping and aggregation, high abundance of ambient RNA, and overall poor suspension quality. If larger samples or other tissues are processed, it is highly recommended to perform trial runs to determine optimal buffer volumes for minimal ambient RNA levels. Nuclei and RNA quality and concentrations need to be examined carefully as overloading results in overall poor performance.
In addition, large amounts of cell debris, causing high levels of ambient RNA not associated with single nuclei influence the sequencing results negatively. Clarification of the nuclei suspension by centrifugation through a sucrose cushion mitigates this problem to some extent, but it can also lead to bias in cell type representation by counter selecting against dense, small nuclei present, for instance, in immune cells16. If this is of concern, the sucrose gradient should be omitted. By contrast, we found that flow cytometry based on DAPI staining was critical to reduce the amount of cell debris in order to produce a high-quality single nuclei suspension.
The isolation of single nuclei has considerable advantages when compared to single-cell approaches8. It is compatible with properly frozen tissue, making the tissue collection more flexible, and circumvents the need of enzyme-based tissue dissociation, which can introduce transcriptional stress responses6,17. Furthermore, it overcomes the dissociation bias that favors the selection of easily dissociable cell types of the renal cortex, which may lead to an underrepresentation of medullary cell types in some enzyme-based approaches5,6,10.
Using a central kidney piece instead of whole kidney tissue further saves resources and corrects for the overrepresentation of abundant cell types as described earlier10. However, depending on the mouse model or phenotype investigated, it may be beneficial to use whole kidney samples instead of a single middle slice. Whole kidney samples may be more representative of true cell proportions, or changes occurring in the whole kidney, whereas a trimmed middle slice proved advantageous for medullary phenotypes or when sample material was limited. This decision, therefore, is highly user-specific and should be considered carefully.
The authors have nothing to disclose.
We thank the Scientific Genomics Platform at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin for technical support.
JL and KMSO were supported by the German Research Foundation (DFG) Research Training Group GRK 2318 and by Research Unit FOR 2841. KMSO was supported by Collaborative Research Grant 1365. AB was supported by funding from the Gottfried Wilhelm Leibniz Prize of the DFG awarded to NR.
Cell sorter | – | – | For fluorescence-activated cell sorting (FACS); e.g. BD FACSAria Cell Sorter. |
Centrifuge 5810 R | Eppendorf | 5811000015 | |
Countess Cell Counting Chamber Slides | Invitrogen | C10228 | |
Countess II FL Automated Cell Counter | Invitrogen | AMQAF1000 | Needs to contain DAPI light cube to count nuclei. Alternatively, nuclei can be counted manually under fluorescence microscope. |
4′,6-Diamidino-2-phenyl-indol-dihydrochlorid (DAPI) | Biotrend | 40043/b | Stock solution prepared with a concentration of 100 µM. Used for nuclei staining in a final concentration of 2 µM. |
D(+)-Sucrose ≥99.9%, ultrapure DNAse-, RNAse-free | VWR | 0335-500G | |
DNA LoBind Microcentrifuge Tubes (1.5 mL) | Eppendorf | 22431021 | |
Ethanol, 70 % | – | – | |
FACS tubes | pluriSelect | 43-10100-46 | |
KIMBLE Dounce tissue grinder set 2 mL complete | Sigma-Aldrich | D8938-1SET | |
Minisart Syringe Filters 0.2 µm | Sartorius | 16534-GUK | |
Nuclease-free Water | Invitrogen | AM9937 | |
Nuclei EZ Prep Nuclei Isolation Kit | Sigma-Aldrich | NUC-101 | Nuclei EZ Lysis Buffer (Product No. N3408) needed for buffer preparation. |
PBS (Phosphate-Buffered Saline) 1X without calcium or magnesium | Corning | 21-040-CV | |
Petri dishes, polystyrene 60 mm | Sigma-Aldrich | P5481 | |
Phosphate-Buffered Saline (PBS) with 10% Bovine Albumin | Sigma-Aldrich | SRE0036 | |
pluriStrainer Mini 100 µm | pluriSelect | 43-10100-46 | |
pluriStrainer Mini 20 µm | pluriSelect | 43-10020-40 | |
pluriStrainer Mini 40 µm | pluriSelect | 43-10040-40 | |
Polystyrene Centrifuge Tube (15 mL) | Falcon | 352099 | |
Razor blades | – | – | |
RiboLock RNase Inhibitor (40 U/µL) | Thermo Fisher | EO0384 | |
Ribonucleoside-vanadyl complex | New England Biolabs | S1402S | Follow manufacturer's instructions (https://international.neb.com/products/s1402-ribonucleoside-vanadyl-complex#Product%20Information). Upon use the 200 mM stock solution is reconstituted to a green-black clear solution by incubating at 65 °C. |
RNAlater Stabilization Solution | Invitrogen | AM7020 | |
RNase AWAY | Fisher Scientific | 11952385 |