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

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 volum....

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.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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