Apr 01, 2026
Fresh-Frozen Kidney Nuclei Isolation and Fixed RNA Profiling with 10X Chromium snRNA-seq
- Anna Smith1,
- Morad Malek1,
- Jamie Allen1,
- Melissa Farrow1
- 1Vanderbilt University
- VU Biomolecular Multimodal Imaging Center / Spraggins Research GroupTech. support email: [email protected]
Protocol Citation: Anna Smith, Morad Malek, Jamie Allen, Melissa Farrow 2026. Fresh-Frozen Kidney Nuclei Isolation and Fixed RNA Profiling with 10X Chromium snRNA-seq . protocols.io https://dx.doi.org/10.17504/protocols.io.261ge39bjl47/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: In development
The protocol is developed but still being written and is a work in progress
Created: February 15, 2023
Last Modified: April 01, 2026
Protocol Integer ID: 77078
Keywords: chromium nuclei isolation kit with rnase inhibitor, frozen kidney nuclei isolation, fixed rna profiling with 10x chromium snrna, chromium nuclei isolation kit, isolating nuclei, 10x genomics single cell, isolated nuclei, fixed rna profiling, rnase inhibitor, rna, 10x chromium snrna, nuclei from tissue
Funders Acknowledgements:
NIH
Grant ID: ABCD123456
Abstract
Scope:
Protocol for isolating nuclei using the Chromium Nuclei Isolation Kit with RNase Inhibitor to be used with 10x Genomics Single Cell 3' (v3.1) RNA Sequencing only.
Expected Outcome:
Isolated nuclei from tissues without debris or large clumps.
Guidelines
(10x Chromium Nuclei Isolation Kit with RNase Inhibitor PN-1000494 Sample Prep User Guide | CG000505 | (Rev A), support.10xgenomics.com)
Materials
(Chromium Nuclei Isolation Kit with RNase Inhibitor 16 rxns, PN-1000494)
Plastics
- 2-ml Tubes DNA LoBind Tubes 2.0 ml Eppendorf 022431048
- 15-ml Tubes Corning 15 ml centrifuge tubes Corning CLS430791
- 50-ml Tubes Corning 50 ml centrifuge tubes Corning CLS430829
Kits & Reagents
- 10% BSA Bovine Serum Albumin in DPBS (10%) (alternatively, use MACS BSA Stock Solution) Millipore-Sigma Miltenyi Biotec A1595 130-091-376
- 1X PBS Phosphate-Buffered Saline without Calcium & Magnesium Corning 21-040-CV
- Nuclease-free Water Molecular Grade Nuclease-free Water Thermo Fisher Scientific AM9937
Cell Counting
- Nucleic Acid Staining Fluorescent Dye VitaStain AOPI Staining Solution (alternatively, use Ethidium Homodimer-1 or Trypan blue)
- Cell Counter Countess™ 3 FL Automated Cell Counter Catalog number: AMQAF2000
Equipment
- Vortex
- Refrigerated Centrifuge
Troubleshooting
Safety warnings
1. Wear proper PPE: gloves, lab coat, and disposable sleeves.
2. Properly dispose of blades and other sharps into sharps container and tissue waste into a biohazard bag.
Section tissues
In general, kidney tissues are to be sectioned according to this protocol. dx.doi.org/10.17504/protocols.io.bt8inrue
For this protocol, two sections of between 50-100 µm should be cut from blocks that are approximately (25mm x 12mm +/- 5mm). Place the two sections into the dissociation tube provided with the 10x Chromium Nuclei Isolation Kit and store at -80 °C until use. The minimum section size for isolation is 50 µm. Try to remove as much embedding material from the tissues as possible.
The remainder of the protocol will be carried out with the 10x Chromium Nuclei Isolation Kit with RNase Inhibitor (PN-1000494) in accordance with Sample Prep User Guide [CG000505, Rev A].
Note: According to the 10x Chromium Nuclei Isolation Protocol, the Chromium Nuclei Isolation Kit requires input tissue masses between 3–50 mg.
Single Cell Gene Expression Buffer Preparation
| A | B | C | D | E | |
| Action | Item | 10x PN | Preparation & Handling | Storage | |
| Place on Ice | Lysis Reagent | 2000558 | Vortex, verify no precipitate, and centrifuge briefly. | 4°C | |
| Surfactant A | 2000559 | Vortex, verify no precipitate, and centrifuge briefly. | 4°C | ||
| Debris Removal Reagent | 2000560 | Vortex, verify no precipitate or layering, and centrifuge briefly. | 4°C | ||
| Reducing Agent B | 2000087 | Thaw to room temperature, vortex, verify no precipitate, and centrifuge briefly. | −20°C | ||
| RNase Inhibitor | 2000565 | Centrifuge briefly. | −20°C | ||
| Nuclei Isolation | Pre-chill assembled | Ambient | |||
| Consumables: | Nuclei Isolation | ||||
| Nuclei Isolation | 2000562 | Column(s) and Collection | |||
| Column | Tube(s) on ice. | ||||
| Collection Tube | 2000563 | ||||
| Nuclease-free Water | — | See Buffer Preparation. | Ambient | ||
| 1X PBS | — | See Buffer Preparation. | Ambient | ||
| 10% BSA | — | See Buffer Preparation. | 4°C | ||
| Frozen Tissue Sample | — | See Tips & Best Practices. | −80°C | ||
| Sample Dissociation Tube | 2000564 | Pre-chill on dry ice. | Ambient | ||
| Obtain | Pestles | 2000561 | Keep on lab bench. | Ambient | |
| Nucleic Acid Staining Fluorescent Dye | — | See Tips & Best Practices. | 4°C | ||
| Vortex | — | See Nuclei Isolation Protocol. | — |
Prepare the following Lysis and Debris Removal Buffers on ice shortly before starting the nuclei isolation protocol. Prepare large volumes in a 15-ml or 50-ml conical tube. Vortex briefly before use.
Lysis Buffer
| A | B | C | D | E | |
| Lysis Buffer (500 µl/rxn) Add reagents in the order listed | PN | 1X+10% (µl) | 4X + 10% (µl) | 8X + 10% (µl) | |
| Lysis Reagent | 2000558 | 550 | 2,200 | 4,400 | |
| Reducing Agent B | 2000087 | 0.55 | 2.2 | 4.4 | |
| Surfactant A | 2000559 | 5.5 | 22 | 44 | |
| Total | – | 556.05 | 2,224.2 | 4,448.4 |
Debris Removal Buffer
| A | B | C | D | E | |
| Debris Removal Buffer (500 µl/rxn) Add reagents in the order listed | PN | 1X+10% (µl) | 4X + 10% (µl) | 8X + 10% (µl) | |
| Debris Removal Reagent | 2000560 | 550 | 2,200 | 4,400 | |
| Reducing Agent B | 2000087 | 0.55 | 2.2 | 4.4 | |
| Total | – | 550.55 | 2,202.2 | 4,404.4 |
Buffer Preparation: Wash and Resuspension Buffer
Prepare the following Wash and Resuspension Buffer on ice shortly before starting the nuclei isolation protocol. Prepare large volumes in a 15-ml or 50-ml conical tube. Vortex briefly before use.
Wash and Resuspension Buffer
| A | B | C | D | E | |
| Wash and Resuspension Buffer (3 ml/rxn) Add reagents in the order listed | PN | 1X+10% (µl) | 4X + 10% (µl) | 8X + 10% (µl) | |
| 1X PBS | 2887.5 | 11550 | 23100 | ||
| 10% BSA | 330 | 1320 | 2640 | ||
| RNase Inhibitor | 2000087 | 82.5 | 330 | 660 | |
| Total | – | 3300 | 13200 | 26400 |
Nuclei Isolation Process
Tissue Dissociation
a. Pre-chill centrifuge to 4°C and place reagents and tubes on ice as indicated in section 2. Label tops and sides of tubes, as well as tops of spin columns, before starting protocol.
b. Prepare Single Cell Gene Expression buffers according to Buffer Preparation section and place on ice.
c. Place Sample Dissociation Tube(s) on dry ice.
d. Obtain frozen tissue sample(s) and place immediately on dry ice.
e. Transfer frozen tissue (3–50 mg) to pre-chilled Sample Dissociation Tube.
f. Transfer Sample Dissociation Tubes(s) to wet ice. Add 200 µl Lysis Buffer to Sample Dissociation Tube. Dissociate tissue with plastic pestle until homogeneous. For multiple samples, add Lysis Buffer to each tissue and then proceed to dissociate one at a time.
g. Add 300 µl Lysis Buffer. Pipette mix 10x. If pipette tip clogs with unhomogenized tissue, continue to dissociate tissue with the pestle until able to pipette mix.
10m
Nuclei Isolation & Cleanup
h. Incubate on ice for 10 min
i. Pipette dissociated tissue into pre-chilled Nuclei Isolation Column assembled with Collection Tube using pipette set to 500 µl. Transfer all liquid from Dissociation Tube to Nuclei Isolation Column to avoid nuclei loss.
j. Centrifuge at 16,000 rcf for 20 secs at 4°C.
k. Discard column. Flowthrough in the Collection Tube will contain nuclei. Vortex 10 sec at 3,200 rpm or max speed to resuspend nuclei. Flowthrough may appear opaque or cloudy. This is normal and it is safe to proceed.
l. Centrifuge Collection Tube for 3 min at 500 rcf at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µl) of supernatant if nuclei pellet is not apparent.
m. Resuspend nuclei pellet in 500 µl Debris Removal Buffer. Gently pipette mix at least 15x, continuing until no pellet can be visualized.
n. Centrifuge at 700 rcf for 10 min at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 µl) of supernatant if nuclei pellet is not apparent.
o. Resuspend nuclei pellet in 1 ml of Wash and Resuspension Buffer.
p. Centrifuge at 500 rcf for 5 min at 4°C. Carefully discard supernatant using a pipette without disturbing nuclei pellet. Leave behind a small fraction (~200 μl) of supernatant if nuclei pellet is not apparent.
q. Resuspend nuclei pellet in 1 ml of Wash and Resuspension Buffer.
r. Centrifuge at 500 rcf for 5 min at 4°C. Carefully discard as much supernatant as possible using a pipette without disturbing nuclei pellet. Leave behind a small remaining volume if the pellet is not visible.
s. Resuspend nuclei pellet in 150-500 µl Wash and Resuspension Buffer, depending on expected recovery for input tissue type and mass. Gently pipette mix 15x using an appropriate pipette for resuspension volume.
45m
t. Vortex nuclei for 3 sec at 3,200 rpm or max speed immediately prior to counting to ensure accurate nuclei count. Pulse spin the tube after vortexing to collect liquid at bottom of tube. DO NOT pulse spin the tube for more than 1 second to ensure that nuclei do not pellet at the bottom of the tube.
u. Determine nuclei concentration using Trypan Blue stain 0.4% using a Countess™ 3 FL Automated Cell Counter and dilute if necessary for target nuclei load. Adjust nuclei concentration as necessary for intended downstream assay.
v. Vortex nuclei for 3 sec at 3,200 rpm or max speed. Pulse spin the tube after vortexing to collect liquid at bottom of tube. DO NOT pulse spin the tube for more than 1 second to ensure that nuclei do not pellet at the bottom of the tube.
w. Keep samples on ice and proceed immediately to 10x Genomics Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling [CG000478, Rev D]
Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling [CG000478, Rev D]
Fix cells using Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, 16 rxns (PN-1000414) and proceed to Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples [CG000527]
Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples [CG000527, Rev E]
Samples undergo probe hybridization, GEM generation and barcoding, GEM recovery and pre-amplification, and fixed RNA gene expression library construction according to the protocol with Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 16 BC (PN-1000476), Chromium Next GEM Chip Q Single Cell Kit, 16 rxns (PN-1000422), and Dual Index Kit TS Set A, 96 rxns (PN-1000251).
Protocol references