Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation
Published: April 30, 2023
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 m…
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.
Açıklamalar
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 |
Etiketler
Bu Makaleden Alıntı Yapın
Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation. J. Vis. Exp. (Pending Publication), e20617, doi: (2023).