Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation
Abstract
Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
In this video, we describe a simple and efficient protocol using gradient centrifugation to isolate single nuclei. Once isolated, these nuclei can be used for sequencing and genome analysis.
Protocol
1. Gradient Centrifugation
- Add 200 µL of 50% iodixanol solution (Table 1) to give a final concentration of 25% iodixanol. Mix well 10 times with a pipette set on 300 mL.
- Add 300 µL of 29% iodixanol solution (Table 2) under the 25% mixture. Use a P1000 fine tip to avoid mixture of the layers.
- Add 300 µl of 35% iodixanol solution (Table 3) under the 29% mixture. Use a P1000 fine tip to avoid mixture of the layers.
CAUTION: This step requires gradual removal of the pipette tip during pipetting to avoid excessive volume displacement. - Place the samples in a swinging bucket centrifuge, spin for 20 min at 3,500 x g at 4°C with the brake off.
- Gently remove the samples without shaking and observe under light. A clear white band of 95% pure nuclei should be visible between the second and third layer (Figure 1).
2. Isolation of Nuclei
- Aspirate the top layers until the white nuclei band at the interphase of 29%–35%.
- Collect the nuclei band in a 200 mL volume, transfer to a fresh tube, and filter with a 20 µm filter (Table of Materials).
NOTE: The nuclei do not need to be resuspended prior to filtration. - Check under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need to be round, and the nuclear membrane should not be distorted.
- Count nuclei using Trypan blue staining on a hemocytometer and aliquot nuclei for snRNA-seq/snATAC-seq.
Table 1: Preparation of 6x Homogenization Buffer Stable Master Mix
| 6x Homogenization Buffer Stable Master Mix | ||
| Reagent | Final Conc. | Vol for 100 (mL) |
| 1 M CaCl₂ | 30 mM | 3.0 |
| 1 M Mg(Ac)₂ | 18 mM | 1.8 |
| 1 M Tris pH 7.8 | 60 mM | 6.0 |
| H₂O | 89.2 | |
| Keep at room temperature, avoid direct exposure to light | ||
Table 2: Preparation of 1 M sucrose
| 1 M Sucrose |
| 34.23 g of sucrose |
| Dissolve in 78.5 mL of water |
| Fill up to 100 mL with water |
Table 3: Preparation of 6x Homogenization Buffer Unstable Solution (650 µL per sample)
| 6x Homogenization Buffer Unstable Solution (650 mL per sample) | ||
| Reagent | Final Conc. | Vol per sample (µL) |
| 6x Homogenization Buffer Stable | 6x | 648.84 |
| 100 mM PMSF (Phenylmethylsulfonyl fluoride) | 0.1 mM | 1.08 |
| 14.3 M β-mercaptoethanol | 1 mM | 0.08 |
Representative Results
Figure 1: Flow chart for nuclei isolation. The flow chart provides a brief outlook on the steps involved in the isolation of single nuclei from a fresh frozen glioma tissue. Representative images for the tumor sample and the nuclear band after Iodixanol/sucrose gradient (circled with red dotted line) are shown.
Disclosures
The authors have nothing to disclose.
Materials
| EDTA (0.5 M) | Thermo Scientific | R1021 | |
| Falcon 15 mL Conical Centrifuge Tubes |
Fisher Scientific | 352096 | |
| Iodixanol (aka Optiprep) | Stem cell technologies | 07820 | |
| NP-40 | Abcam | ab142227 | |
| Safe lock tubes 1.5 mL | Eppendorf | 0030120086 | |
| Safe lock tubes 2.0 mL | Eppendorf | 0030120094 | |
| Sucrose | Sigma | S0389 | |
| Wide Bore pipette tips (1000 µL) | Themo Fisher Scientific | 2079GPK | |
| Wide Bore pipette tips (200 µL) | Themo Fisher Scientific | 2069GPK |
