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EoE Sub Articles

1. Gradient Centrifugation
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	<li>Add 200 &#181;L 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.</li>
	<li>Add 300 &#181;L of 29% iodixanol solution (Table 2) under the 25% mixture. Use a P1000 fine tip to avoid mixture of the layers.</li>
	<li>Add 300 &#181;l of 35% iodixanol solution (Table 3) under the 29% mixture. Use a P1000 fine tip to avoid m...

gradient centrifugation based purification of nuclei: a technique to obtain ultrapure fraction of intact nuclei using density gradient centrifugation

Source: Narayanan, A. et al. <a href="https://www.jove.com/v/61542/nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus">Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/20617/20617_Figure1.jpg" /> 
Figure 1: Flow chart for nuclei isolation.&#160;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.

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

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, ...

To isolate and purify intact nuclei, begin by taking a crude nuclei fraction in a tube. The fraction contains intact nuclei along with broken nuclei fragments and debris.
Mix this fraction with a suitable density gradient medium such that it forms a suspension of the lowest concentration.
Now, layer solutions of increasing densities of the same gradient medium beneath the pre-prepared crude nuclei fraction to generate a multi-step discontinuous density gradient. This step allows the crude nuclei fraction to form a band at the top of the gradient.
Next, centrifuge the tube at a low speed.
Based on the differences in shape and mass, intact nuclei migrate differently compared to debris and nuclear fragments. This results in intact nuclei forming a separate band.
After centrifugation, locate the distinct band consisting of intact nuclei at the interface of density gradient solutions.
Using a pipette, aspirate the top layers of the gradient to facilitate access to the pure nuclei band. Collect the nuclei-containing band into a fresh tube.
Visualize these nuclei under a light microscope. Intact nuclei appear perfectly round with an intact nuclear membrane surrounding them.
Finally, store the pure nuclear fraction for further analysis.

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

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