Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation

Samenvatting

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

1. 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.
2. Add 300 µL of 29% iodixanol solution (Table 2) under the 25% mixture. Use a P1000 fine tip to avoid mixture of the layers.
3. 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.
4. Place the samples in a swinging bucket centrifuge, spin for 20 min at 3,500 x g at 4°C with the brake off.
5. 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

1. Aspirate the top layers until the white nuclei band at the interphase of 29%–35%.
2. 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.
3. 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.
4. 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

Resultaten

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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
EDTA (0.5 M)	Thermo Scientific	R1021
Falcon 15 mL Conical Centrifuge<br/> 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 &micro;L)	Thermo Fisher Scientific	2079GPK
Wide Bore pipette tips (200 &micro;L)	Thermo Fisher Scientific	2069GPK