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

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

Add 200 microliters of 50% iodixanol solution to the nuclei for a final concentration of 25% iodixanol and mix 10 times with the pipette set to 300 microliters.
Then, add 300 microliters of 29% iodixanol solution under the 25% mixture using a P1000 fine tip to avoid mixing the layers. Add 300 microliters of 35% iodixanol solution under the 29% mixture with a P1000 fine tip. Then, slowly remove it.
Place the samples in a swinging bucket centrifuge and spin them for 20 minutes at 3,500 x g at 4 degrees Celsius with the brake off.
Gently remove the samples without shaking and observe them under light. A clear white band of 95% pure nuclei should be visible between the second and third layer.
To isolate the nuclei, aspirate the top layers to expose the white nuclei band at the interface. Collect the nuclei band in a 200-milliliter volume and transfer it to a fresh tube. Then, filter it with a 20-micrometer filter.
Check the nuclei under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. They should be round, and the nuclear membrane should not be distorted.

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1. Gradient Centrifugation
<ol>
	<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 mixture of the layers. 
	CAUTION: This step requires gradual removal of the pipette tip during pipetting to avoid excessive volume displacement.</li>
	<li>Place the samples in a swinging bucket centrifuge, spin for 20 min at 3,500 x&#160;g&#160;at 4&#176;C with the brake off.</li>
	<li>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).</li>
</ol>
2. Isolation of Nuclei
<ol>
	<li>Aspirate the top layers until the white nuclei band at the interphase of 29%&#8211;35%.</li>
	<li>Collect the nuclei band in a 200 mL volume, transfer to a fresh tube, and filter with a 20 &#181;m filter (Table of Materials). 
	NOTE: The nuclei do not need to be resuspended prior to filtration.</li>
	<li>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.</li>
	<li>Count nuclei using Trypan blue staining on a hemocytometer and aliquot nuclei for snRNA-seq/snATAC-seq.</li>
</ol>
Table 1: Preparation of 6x Homogenization Buffer Stable Master Mix
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">6x Homogenization Buffer Stable Master Mix</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Final Conc.</td>
			<td>Vol for 100 (mL)</td>
		</tr>
		<tr>
			<td>1 M CaCl&#8322;</td>
			<td>30 mM</td>
			<td>3.0</td>
		</tr>
		<tr>
			<td>1 M Mg(Ac)&#8322;</td>
			<td>18 mM</td>
			<td>1.8</td>
		</tr>
		<tr>
			<td>1 M Tris pH 7.8</td>
			<td>60 mM</td>
			<td>6.0</td>
		</tr>
		<tr>
			<td>H&#8322;O</td>
			<td></td>
			<td>89.2</td>
		</tr>
		<tr>
			<td colspan="3">Keep at room temperature, avoid direct exposure to light</td>
		</tr>
	</tbody>
</table>


Table 2: Preparation of 1 M sucrose
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M Sucrose</td>
		</tr>
		<tr>
			<td>34.23 g of sucrose</td>
		</tr>
		<tr>
			<td>Dissolve in 78.5 mL of water</td>
		</tr>
		<tr>
			<td>Fill up to 100 mL with water</td>
		</tr>
	</tbody>
</table>


Table 3: Preparation of 6x Homogenization Buffer Unstable Solution (650 &#181;L per sample)

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">6x Homogenization Buffer Unstable Solution (650 mL per sample)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Final Conc.</td>
			<td>Vol per sample (&#181;L)</td>
		</tr>
		<tr>
			<td>6x Homogenization Buffer Stable</td>
			<td>6x</td>
			<td>648.84</td>
		</tr>
		<tr>
			<td>100 mM PMSF (Phenylmethylsulfonyl fluoride)</td>
			<td>0.1 mM</td>
			<td>1.08</td>
		</tr>
		<tr>
			<td>14.3 M &#946;-mercaptoethanol</td>
			<td>1 mM</td>
			<td>0.08</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15 mL Conical Centrifuge 
			Tubes</td>
			<td>Fisher Scientific</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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.

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

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