Apr 28, 2026
Nuclei isolation from zebrafish larvae using 10x genomics nuclei isolation kit
- Neelakanteswar Aluru1,
- Christopher S. Murray1
- 1Biology department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
Protocol Citation: Neelakanteswar Aluru, Christopher S. Murray 2026. Nuclei isolation from zebrafish larvae using 10x genomics nuclei isolation kit. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn5k6mg5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2026
Last Modified: April 28, 2026
Protocol Integer ID: 315827
Keywords: nuclei isolation from zebrafish larvae, using 10x genomics nuclei isolation kit single, 10x genomics nuclei isolation kit, 10x genomics nuclei isolation kit single, such as the 10x genomics nuclei isolation kit, optimized nuclei isolation protocol, ambient rna contamination, nuclei isolation protocol, protocol for nuclei isolation, zebrafish larvae, including zebrafish, nuclei isolation, nucleus rna, zebrafish, nuclei integrity, profiling gene expression, sequencing experiment, larvae, nuclei with suitable morphology, nuclei, gene expression in complex tissue
Funders Acknowledgements:
National Institutes of Health
Grant ID: P01ES028938 and R21ES034839
National Science Foundation
Grant ID: OCE-2418297
Disclaimer
This protocol reflects how we currently process zebrafish larvae. While we have optimized the workflow, further improvements are still needed to minimize ambient RNA contamination, and these will be incorporated as they are optimized.
Abstract
Single-nucleus RNA sequencing (snRNA-seq) has become a powerful approach for profiling gene expression in complex tissues, yet optimized nuclei isolation protocols are often limited to mammalian systems. Commercial kits, such as the 10x Genomics Nuclei Isolation Kit, provide standardized workflows but are not validated for many non-mammalian models, including zebrafish. Although individual laboratories have begun adapting these protocols for zebrafish, detailed step-by-step methodological descriptions are often not reported in publications limiting reproducibility and broader adoption.
Here, we present an optimized protocol for nuclei isolation from zebrafish (Danio rerio) larvae at 5–6 days post-fertilization (dpf), adapted from the manufacturer’s guidelines with targeted modifications to improve yield and nuclei integrity. The optimized workflow produces nuclei with suitable morphology and concentration for downstream snRNA-seq applications, and we demonstrate its successful implementation in a sequencing experiment. Despite these improvements, ambient RNA contamination remains a limitation, highlighting the need for further refinement. This protocol provides a practical and reproducible starting point for researchers working with zebrafish larvae and will be updated as additional optimizations are developed to further enhance sample quality.
Materials
1. 10x Nuclei isolation kit (Catalog # PN-1000494)
2. 1X Phosphate buffered saline (PBS; Fisher Scientific catalog # MT21040CV)
3. 10% Bovine Serum Albumin (BSA, Sigma catalog # A1595)
4. Bel-Art™ Flowmi™ Cell Strainers for 1000µL Pipette Tips (Fisher Scientific catalog # 14-100-150)
5. 2 mL Lo-bind Eppendorf tubes (Eppendorf catalog # 022431048)
6. 0.6 mL Eppendorf tubes (USA Scientific)
7. Ice bucket
8. Pipettes, filter pipette tips
9. LUNA FX7 Automated cell counter (Logos biosystems, Annandale, VA)
10. LUNA FX7 Ultra-low Fluorescence disposable counting slide (Logos biosystems # L12005)
11. Acridine Orange/Propidium Iodide stain (Logos biosystems # F23001)
12. Fluorescent upright microscope
13. MedOne C-Chip™ Disposable Hemacytometer (Fisher scientific catalog # 22-600-113)
Before start
1. Install the swinging bucket rotor (Eppendorf S24-11AT) in the Eppendorf 5430R centrifuge. A swinging bucket rotor is preferred; however, a standard fixed-angle rotor can also be used.
2. Pre-cool the refrigerated centrifuge to 4°C.
3. Thaw the 10x Genomics Nuclei Isolation Kit reagents stored at −20°C on ice. Ensure that all components are fully thawed and free of precipitate before use.
4. Prepare lysis buffer, debris removal buffer, and wash and resuspension buffer as per the kit instructions and keep them on ice. The following volumes are for isolating nuclei from 8 samples. Corresponding volumes for a single sample are also provided to facilitate scaling to any number of samples. However, we do not recommend scaling beyond 8 samples in order to preserve the nuclei integrity.
a) Lysis buffer
| Item | Volume (1X + 10%) | Volume (8X + 10%) |
|------|------------------|-------------------|
| Lysis reagent | 550 µL | 4.4 mL |
| Reducing agent B | 0.55 µL | 4.4 µL |
| Surfactant A | 5.5 µL | 44.0 µL |
b) Debris Removal Buffer
| Item | Volume (1X + 10%) | Volume (8X + 10%) |
|------|------------------|-------------------|
| Debris Removal reagent | 550 µL | 4.4 mL |
| Reducing agent B | 0.55 µL | 4.4 µL |
c) Wash and Resuspension Buffer
| Item | Volume (1X + 10%) | Volume (8X + 10%) |
|------|------------------|-------------------|
| 1X PBS | 1.925 mL | 11.55 mL |
| 10% BSA | 220 µL | 1.32 mL |
| RNase inhibitor | 55 µL | 330 µL |
Isolation procedure
Quickly thaw samples from −80°C on ice and immediately add 200 µL of lysis buffer.
Using the plastic pestle provided in the kit, homogenize the sample on ice by gently twisting the pestle for 20–30 seconds. Visually confirm that the larvae are fully disrupted and that the homogenate appears uniformly cloudy.
Add the remaining 300 µL of lysis buffer and gently mix.
Incubate the sample on ice for 10 minutes.
Filter the homogenate through a 40 µm Flowmi™ strainer into a new pre-chilled Eppendorf tube. If the strainer becomes clogged, replace it with a new one and continue filtering the remaining sample.
Transfer the filtered homogenate into the column placed inside a 2 mL Eppendorf tube on ice. It is recommended to remove (cut) the tube caps before inserting the columns to avoid issues during centrifugation. Make a note of the orientation of the tube to help identify the pellet after centrifugation.
Carefully place the tubes in a swinging bucket rotor and ensure that the tubes are properly balanced.
Centrifuge to pellet nuclei. Although 10x Genomics recommends 16,000 × g, we found this resulted in increased debris in the final isolate. A speed of 3,000 × g for 20 seconds was sufficient to effectively pellet nuclei while reducing debris.
Carefully remove the supernatant without disturbing the pellet, leaving a small volume behind to avoid disruption.
If the column becomes clogged, transfer the homogenate to a new column and combine the flowthroughs. While not ideal, this can help preserve nuclei, especially when working with small samples.
Discard the column. Gently mix the flowthrough in the collection tube for ~10 seconds to resuspend any pelleted nuclei.
Centrifuge the resuspended nuclei at 1,000 × g for 3 minutes. At this and all subsequent steps, carefully note the position of the pellet. Remove the supernatant gently by pipetting, leaving a small volume to avoid disturbing the pellet.
Add 500 µL of Debris Removal Buffer and resuspend the pellet by gentle pipetting.
Centrifuge at 1,000 × g for 5 minutes. Carefully remove the supernatant without disturbing the pellet.
Add 500 µL of Wash and Resuspension Buffer and centrifuge at 1,000 × g for 5 minutes.
Discard the supernatant. The nuclei pellet may be washed once more; however, if the pellet is very small, a single wash is acceptable to minimize sample loss.
Resuspend the nuclei pellet in 50–100 µL of Wash and Resuspension Buffer by gentle pipetting. Keep the sample on ice and proceed to nuclei counting using AO/PI.
Nuclei counting using acridine orange (AO) and propidium iodide (PI)
Mix 2 µL of AO/PI staining solution with 18 µL of the nuclei suspension in a new tube. Incubate for 1–2 minutes at room temperature.
Load 20 µL of the stained sample onto a counting slide, taking care to avoid introducing air bubbles.
Insert the slide into the automated cell counter.
Use the default AO/PI settings provided by the instrument.
Note that these counters are optimized for whole-cell viability assessments. When applied to nuclei, the output can be misleading, as nuclei are classified as “dead cells” due to PI staining. In this context, these dead cells should be interpreted as nuclei rather than non-viable cells. At this stage there should be no live cells in the samples.
For example, here are the results of one of our sample – Total cells 3.95E06 cells/mL, Live cells 3.86E04 cells/mL and Dead cells 3.96E06 cells/mL. The dead cells are indeed live nuclei.
Visualization of nuclei
Any upright fluorescence microscope can be used to assess nuclei morphology. We used the Thermo Scientific EVOS M3000 Imaging System equipped with GFP and RFP filter cubes.
Prepare 20 µL of nuclei suspension stained with AO/PI as described above, and load 10 µL onto a hemocytometer slide. Under these conditions, intact cells (if present) will fluoresce in the GFP channel (AO), while nuclei will be visualized in the RFP channel (PI).
Nuclei can be counted at 10X or 20X magnification under brightfield settings and compared with counts obtained from automated cell counters.
Examine the sample at 40X magnification to confirm that nuclei exhibit smooth, intact morphology with minimal debris or aggregation.
Acknowledgements
We acknowledge the support of Dr. Alicia Vaca and Ms. Chelsea Otis at the Broad Institute, Boston, for their assistance with troubleshooting the protocol. We also thank Dr. Nicole Eckart at 10x Genomics and Ms. Ria Massoni at the Marine Biological Laboratory, Woods Hole, for their helpful suggestions.
**Funding**
This work was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award numbers P01ES028938 and R21ES034839. Additional support for this research was provided by the National Science Foundation under award number OCE-2418297. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
**Funding**
This work was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award numbers P01ES028938 and R21ES034839. Additional support for this research was provided by the National Science Foundation under award number OCE-2418297. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
**Funding**
This work was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award numbers P01ES028938 and R21ES034839. Additional support for this research was provided by the National Science Foundation under award number OCE-2418297. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.