Name: nuclei isolation from whole tissue using a detergent and enzyme-free method
Uploaded: 2020-06-24T14:00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method at JoVE.com

We thank members of the Dr. Sabine Costagliola and Singh lab for comments on the manuscript. This work was supported by the Fonds de la Recherche Scientifique-FNRS under Grant number 34772792 &#8211; MISU to S.P.S.

All the procedures presented below were performed in accordance with institutional (Universite&#769; Libre de Bruxelles (ULB)) and national ethical and animal welfare guidelines and regulation, which were approved by the ethical committee for animal welfare (CEBEA) from the Universite&#769; Libre de Bruxelles (protocols 578N-579N).
1. Preparation before tissue dissection
<ol>
	<li>Prepare 0.2% Tricaine solution in PBS for euthanizing the zebrafish. Chill the solution on ice.</li>
	<li>Prepare a 30 mm Petri dish for mincing the tissue.</li>
	<li>Prepare ice cold 1x PBS (10 mL per tissue sample).</li>
	<li>Cool the centrifuge to 4 &#176;C.</li>
	<li>For nuclei isolation, use a detergent-free nuclei isolation kit (Table of Materials).</li>
	<li>Before starting the protocol, pre-chill buffer A and B provided in the kit by placing them on ice for at least 30 min prior to nuclei isolation.</li>
	<li>For handling the isolated nuclei, coat the plastic reagents such as tubes and pipette tips with 5% BSA solution. For this, prepare the solution by dissolving 2 g of BSA in 40 mL of PBS. Coating plastic items with 5% BSA reduces nuclei sticking to plastic. This step enhances the recovery of isolated nuclei.</li>
	<li>Coat the pipette tips by pipetting 5% BSA solution 2-3 times. Air-dry the tips for 2 hours. Prepare 10 plastics tips per sample.</li>
	<li>Coat the tubes for collection of nuclei by filling them with 5% BSA. Invert the tubes 3 times to ensure an efficient coating. Remove the solution and air-dry the tubes upside down on a clean tissue paper for 2 hours. Per sample, coat one collection tube provided in the kit. Additionally, prepare coated 1.5 mL tubes for nuclei collection after FACS. 
	NOTE: Glass tips are highly recommended alternative to the plastic pipette tips to minimize the sticking.</li>
</ol>
2. Dissection of zebrafish brain
<ol>
	<li>For euthanizing the zebrafish, prepare a 90 mm Petri dish with 25 mL of ice-cold Tricaine solution.</li>
	<li>Carefully, take the zebrafish from the tank using a fishing net and place it into the Petri dish.</li>
	<li>Euthanize the fish by leaving it in Tricaine for 5 min.</li>
	<li>Decapitate the animal with a sharp razor blade.</li>
	<li>Using forceps, gently break open the skull and remove soft tissues, skin and bones from ventral and dorsal side of the skull.</li>
	<li>Gently, transfer the brain into a fresh 30 mm dish containing ice cold PBS.</li>
	<li>Mince the brain into small pieces using a razor blade to ease the loading of the sample on the spin column.</li>
</ol>
3. Single nuclei isolation
<ol>
	<li>Transfer the minced tissue to the filter cartridge provided in the nuclei isolation kit and add 200 &#181;L of cold buffer A to sensitize the tissue. Grind the tissue using the plastic rod provided by the kit for 2 min.</li>
	<li>Add 300 &#181;L of cold buffer A and incubate the filter cartridge on ice with cap open for 10 min.</li>
	<li>Cap the cartridge and resuspend the tissue by inverting the tube 5 times.</li>
	<li>Centrifuge at 16,000 x g for 30 s. In this step, cells are ruptured when passing through the filter and high-speed centrifugal force is applied. The flow through contains intact nuclei, which pellet at the bottom of the tube.</li>
	<li>Discard the filter and resuspend the pellet by vortexing vigorously for 10 s.</li>
	<li>Pellet the nuclei by centrifuging the solution at 500 x g for 3 min. Discard the supernatant carefully as the nuclei pellet is colorless.</li>
	<li>Resuspend the pellet in 0.8 mL of cold buffer B and centrifuge at 600 x g for 10 min. In this step, nuclei are separated from membrane debris. The colorless pellet obtained contains isolated nuclei.</li>
	<li>Resuspend the isolated nuclei in 500 &#181;L of PBS with 5% BSA. Keep the nuclei suspension on ice to perform FACS after the quantification.</li>
</ol>
4. Visualization of nuclei morphology
<ol>
	<li>Confirm nuclear morphology by Hoechst staining. For this, remove 100 &#181;L of single nuclei suspension in a new tube using BSA coated tips. To stain the nuclei, add 0.1 &#181;L of Hoechst (1 mg/mL). Gently vortex the tube.</li>
	<li>Transfer the nuclei suspension to glass bottom dish for imaging.</li>
	<li>Image the nuclei using a fluorescence microscope with laser excitation settings of ~405 nm (Violet) wavelength.</li>
</ol>
5. FACS based enrichment of nuclei
<ol>
	<li>Before performing FACS, filter the nuclei using a 40 &#181;m cell strainer into BSA coated tube.</li>
	<li>Dilute the filtered suspension by adding PBS with 5% BSA to a final volume of 1,000 &#181;L.</li>
	<li>Label two round bottom FACS tube as &#8216;control&#8217; and &#8216;stained&#8217;. The &#8216;control&#8217; tube will contain un-stained nuclei, while the &#8216;stained&#8217; tube will have Hoechst stained nuclei.</li>
	<li>Transfer 250 &#181;L of the nuclei suspension into &#8216;control&#8217; tube using BSA coated pipette tip.</li>
	<li>Transfer the remaining 750 &#181;L solution to FACS tube labeled as &#8216;stained&#8217; and add 1 &#181;L of Hoechst dye to stain the nuclei. Mix by slow vortexing.</li>
	<li>Load the unstained control sample to cell sorter. Record 5000 events.</li>
	<li>Load the stained samples and record 5000 events.</li>
	<li>Draw FACS gates that allows identification of single nuclei. Nuclei can be selected by comparing the Hoechst fluorescence signal between control and stained sample.</li>
	<li>Sort Hoechst-positive nuclei from the stained tube into new 1.5 mL tube containing 50 &#181;L of PBS with 5% BSA. 
	NOTE: Isolated nuclei can be collected into a desired medium according to requirements of the downstream application.</li>
</ol>

     

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The authors have no conflict of interest. The payment for making the article open access was made by Invent Biotechnologies Inc., USA.

Profiling the transcriptome and epigenome at a single-cell resolution has revolutionized the study of biological systems. Studies at the resolution of a single cell for a solid tissue depend on dissociation of the organ into individual cells or nuclei. Dissociation is a destructive procedure that can introduce technical artifacts, which can prevent development of an accurate representation of the system5,6. For instance, enzymatic dissociation can harm cells with complex morphologies, such as neurons or podocytes, and can induce expression of stress and heat-shock response genes7,12. Additionally, use of detergent during dissociation can rupture the nuclear membrane and lead to aggregation23,25. Thus, optimizing the dissociation to obtain a single cell or nuclei suspension of the highest quality is paramount to the success of high-throughput profiling experiments.
Here, we demonstrate a detergent and enzyme-free nuclei isolation method that allows extraction of intact nuclei from zebrafish brain in less than 20 minutes. The protocol yields nuclei with typical morphology and robust integrity (Figure 2). From a single zebrafish brain weighing 6 mg, the protocol yields a total of 60,000 nuclei determined by a hemocytometer count. The isolated nuclei can be utilized for multiple downstream applications, including snRNA-seq, ATAC-seq and immunostaining. The isolated nuclei may include cross-contamination from cytoplasmic fractions, particularly from components of endoplasmic reticulum and mitochondria. For high-throughput profiling experiments, clearance of cellular debris, particularly mitochondria, is strongly recommended. Flow cytometry (Figure 3) provides a viable option for purification of nuclei. Alternatively, the sucrose gradient can also be utilized for removal of debris.
The protocol has been tested on mouse thyroid gland (data not shown) and provides results similar to zebrafish brain tissue. Overall, the protocol provides a robust, reproducible, and universal method for preparation of single nucleus suspension, helping to simplify logistics for high-throughput profiling experiments.

Single-cell RNA-seq (scRNA-Seq) and ATAC-seq are versatile tools to study complex biological systems at single-cell resolution. They are widely utilized to define cell subtypes and states, gene networks and to assess cellular heterogeneity. A prerequisite for performing scRNA-seq is the preparation of a single cell suspension by tissue dissociation. Due to the variation in the extracellular matrix composition and mechanical properties, individual tissues require optimization of the dissociation protocol for preparation of single cell suspension.
Dissociation of tissues into single cells typically involves treatment with digestive enzymes, including collagenase, dispase or trypsin, at 37 &#176;C1,2,3,4. As transcriptional machinery remains active at 37 &#176;C, enzymatic dissociation can introduce mRNA expression artifacts and noise5,6. Notably, prolonged incubation can induce stress responsive genes and heat-shock response in a non-uniform manner &#8211; leading to technical variability in the experiment7.
Another drawback of generating a single cell suspension is the difficulty in obtaining viable and intact cell-types with complex morphologies. In particular, neurons, adipocytes and podocytes are challenging to isolate8,9,10,11. For instance, Wu and colleagues demonstrated the absence of glomerular podocytes in scRNA profiles from an adult mouse kidney12. Similar nonoptimal observations have been made regarding the recovery of interconnected neurons from brain tissue8,13,14. In sum, dissociation protocols can introduce detection bias towards easier to dissociate cell-types, leading to a misrepresentation of the cellular architecture of the organ.
To overcome the technical noise and bias introduced during sample preparation in scRNA-Seq., isolation and profiling the nucleus provides an attractive alternative. As nuclear morphology is similar between different cell-types, isolation of the nuclei circumvents the issue of isolating intact and viable cells with complex morphologies. For instance, Wu and colleagues demonstrated successful profiling of glomerular podocytes with the single-nucleus RNA-Seq. (snRNA-Seq.) of an adult mouse kidney, which was missing from scRNA-Seq12. Intriguingly, comparative studies between single-cell and single-nucleus RNA-seq have suggested a decrease in induction of stress and heat-shock response genes with snRNA-Seq12. The studies further suggest a high correlation between the genes detected by the two methods. However, a recent study on human microglia failed to detect genetic activation in Alzheimer&#8217;s disease15. Thus in certain contexts, snRNA-Seq is a suitable alternative for scRNA-Seq16,17. Additionally, the nuclear isolation can be utilized for single-cell ATAC-Seq., providing information about the regions of open-chromatin within individual cells.
The protocol for nuclei isolation involves three major steps: i) detergent-based lysis of cell membrane to release the nucleus; ii) tissue homogenization using a Dounce homogenizer; and iii) enrichment of nuclei and removal of cell debris using gradient centrifugation or flow cytometry18,19,20,21,22. Among this, the first two steps depend on the tissue type and need to be empirically optimized. Mild detergent leads to partial rupture of cell membrane and inefficient retrieval of nuclei from the tissue23. On the other hand, high level of detergent and harsh homogenization leads to rupture of the nuclear membrane and their loss24,25. Ruptured nuclei further tend to clump together and form aggregates, which if not removed can lead to artifacts in the downstream profiling experiment.
To circumvent the issues related to detergent optimization for nuclei isolation, we introduce a protocol to isolate intact nuclei from fresh samples using a detergent-free and spin-column-based method. The protocol yields nuclei from whole organ within 20 minutes, limiting the induction of artifactual transcription. The isolated nuclei can be enriched with FACS for single-nuclei RNA-Seq. and ATAC-seq, providing a simple and universal method that enables robust and reproducible high-throughput profiling.

<table>
	<tbody>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Carl Roth</td>
			<td>90604-29-8</td>
			<td>Albumin fraction V</td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>FACSAria III</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sartorius</td>
			<td>A-14C</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes (1.5 mL)</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon (15 mL)</td>
			<td>Corning</td>
			<td>352096</td>
			<td>Polypropylene centrifuge tubes</td>
		</tr>
		<tr>
			<td>Falcon (5 mL )</td>
			<td>Corning</td>
			<td>352052</td>
			<td>Polystyrene round bottom test tubes</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmi cell strainer (40 &#956;m)</td>
			<td>Sigma</td>
			<td>BAH136800040</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottle (250 mL)</td>
			<td>VWR</td>
			<td>215-1593</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottomed dish</td>
			<td>World Precision Instruments</td>
			<td>FD3510-100</td>
			<td>Fluorodish 35 mm</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>VWR</td>
			<td>612-1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipette socket</td>
			<td>Carl Roth</td>
			<td>388.1</td>
			<td>Pipetting aid pi-pump 2500</td>
		</tr>
		<tr>
			<td>Hoechst staining dye solution</td>
			<td>Abcam</td>
			<td>ab228551</td>
			<td>Hoechst 33342</td>
		</tr>
		<tr>
			<td>Minute Detergent-Free Nuclei Isolation Kit</td>
			<td>Invent Biotechnologies</td>
			<td>NI-024</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X)</td>
			<td>ThermoFisher</td>
			<td>70011069</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (30 mm)</td>
			<td>FisherScientific</td>
			<td>11333704</td>
			<td>Pyrex</td>
		</tr>
		<tr>
			<td>Petri dish (90 mm)</td>
			<td>Corning</td>
			<td>758-10178-CS</td>
			<td>Gosselin</td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>VWR</td>
			<td>89079</td>
			<td>10 &#956;L, 200 &#956;L, 1000 &#956;L</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Gilson</td>
			<td>F167380</td>
			<td>Pipetman</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Swann-Morton</td>
			<td>7981809</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methane sulfonate</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex machine</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Vortex-Genie 2</td>
		</tr>
	</tbody>
</table>

nuclei isolation from whole tissue using a detergent and enzyme-free method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

--The protocol described above was used to generate single nucleus suspension directly from zebrafish brain tissue. The isolation typically requires 20 minutes and avoid the use of detergent or digestive enzyme. A schematic summarizing the individual steps of the protocol is provided in Figure 1, which can be printed to be used for guidance.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61471/61471fig01.jpg" /> 
Figure 1: Schematic of detergent-free spin-column-based method for nuclei isolation. 
Graphical representation of the individual steps performed during extraction of nuclei from fresh zebrafish brain tissue. <a href="https://www.jove.com/files/ftp_upload/61471/61471fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Visualization of nuclear morphology
For qualitative confirmation of the nuclear morphology, the isolated nuclei were stained with Hoechst and visualized using fluorescence microscopy. The nuclei appeared intact, round and well-separated (Figure 2). Importantly, nuclear aggregation, a sign of nuclear membrane rupture, was absent.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61471/61471fig02.jpg" /> 
Figure 2: Single nuclei isolation from zebrafish brain. 
Fluorescence microscopy image of Hoechst-stained nuclei demonstrating their intact morphology. Scale bar: 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61471/61471fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
FACS-based enrichment of the intact nuclei
Enrichment of isolated nuclei and removal of cellular debris was performed by flow cytometry by gating on the presence of a Hoechst fluorescence signal. The Hoechst signal was detected upon excitation with violet, 405 nm, laser (Brilliant Violet 421 &#8211; BV421). Unstained nuclei displayed background fluorescence (Figure 3A, Supplementary Figure 1A), while stained nuclei exhibited strong fluorescent signal (Figure 3B, Supplementary Figure 1B). As illustrated in Figure 3C, the unstained and Hoechst stained nuclei were well segregated in the violet channel.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61471/61471fig03.jpg" /> 
Figure 3: Isolated nuclei display strong and specific Hoechst fluorescent signal in flow cytometry. 
Histogram plots for single nuclei suspension displaying the distribution of Hoechst staining. Hoechst is excited by violet, 405 nm, laser (Brilliant Violet 421 &#8211; BV421). The unstained sample (A) displays signal in the range of 100-103, while Hoechst stained nuclei (B) emit signal in the 103-105 range. An overlay of fluorescence intensity emitted by unstained (grey) and stained (blue) samples (C) demonstrates clear separation between the two populations. <a href="https://www.jove.com/files/ftp_upload/61471/61471fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61471/61471supfig01v2.jpg" /> 
Supplementary Figure 1: Flow cytometry gating strategy for isolated nuclei. Representative flow plots for isolated nuclei suspension. Isolated nuclei were analyzed using forward scatter and Violet&#160;laser BV421 which excites Hoechst at 405 nm. The unstained sample (A) displayed BV421 signal in the 100-103 range. Out of 13130 events, 141 events were detected as single nuclei based on FSC-A (1.07% of total), and 0 events for unstained nuclei based on BV421 signal (0% of total). The Hoechst stained nuclei (B) displayed BV421 signal in the 103-105 range. Out of 50000 events, 2418 events were detected as single nuclei based on FSC-A (4.84% of total), and 2414 events for Hoechst-positive nuclei based on BV421 signal (4.83% of total). <a href="https://cloudflare.jove.com/files/ftp_upload/61471/61471supfig01.jpg" target="_blank">Please click here to download this figure.</a>

Watch this Scientific Journal Video about Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method at JoVE.com

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Preparing a single cell or single nuclei suspension is a critical step for high throughput transcriptome and epigenome profiling. Our protocol provides a simple, reproducible, and universal method for single nuclei isolation. Contrary to other methods requiring detergent for nuclei isolation, our protocol is column-based, which is detergent and enzyme-free.It enables nuclei isolation in less than 30 minutes. Our protocol simplifies nuclei isolation. It provides a robust method to prepare single nuclei suspension from hard-to-dissociate tissues.To begin, pre-chill buffer A and B from a detergent-free nuclei isolation kit by placing them on ice for at least 30 minutes. Coat tubes and pipette tips with 5%BSA solution, which will enhance recovery of the nuclei. To coat the pipette tips, pipette 5%BSA solution two to three times and air dry them for two hours.To coat the tubes, fill them with BSA and invert them three times. Remove the solution and air dry the tubes upside down on a clean tissue paper for two hours. Coat one collection tube and one 1.5 milliliter tube per sample.Use forceps to gently break open the skull and remove soft tissues, skin, and bones from the ventral and dorsal sides of the skull. Then gently transfer the brain into a 30 millimeter dish containing ice cold PBS and mince the brain into small pieces using a razor blade. To isolate single nuclei, transfer the minced tissue to the filter cartridge provided in the nuclei isolation kit and remove excess solution and add 200 microliters of cold buffer A.Grind the tissue with a plastic rod for two minutes.Add 300 microliters of cold buffer A to the filter cartridge and incubate it on ice with the cap open for 10 minutes. Then cap the cartridge and resuspend the tissue by inverting the tube five times. Centrifuge the sample tube at 16, 000 times g for 30 seconds to rupture the cells.The flow-through contains intact nuclei, which form a colorless pellet at the bottom of the tube. Discard the filter and then resuspend the pellet by vortexing vigorously for 10 seconds. Pellet the nuclei again by centrifuging the solution at 500 times g for three minutes.Then carefully discard the supernatant. Resuspend the pellet in 0.8 milliliters of cold buffer B by pipetting two to three times and centrifuge the nuclei at 600 times g for 10 minutes, which will separate them from the membrane debris. Carefully discard the supernatant.Resuspend the isolated nuclei in 500 microliters of PBS with 5%BSA and keep the suspension on ice until FACS. To confirm nuclear morphology with HEX staining, transfer 100 microliters of the single nuclei suspension to a new tube using the BSA coated tips. Stain the nuclei by adding 0.1 microliter of HEX and gently vortexing the tube.Transfer the nuclei suspension to a glass bottom dish and image of the nuclei using a fluorescence microscope with a laser excitation setting of 405 nanometers. Before performing FACS, filter the nuclei with a 40 micrometer cell strainer into a BSA coated tube. Dilute the filtered suspension by adding PBS with 5%BSA to a final volume of 1, 000 microliters.Label two round bottom FACS tubes as control and stained and transfer 250 microliters of the nuclei suspension into the control tube using a BSA coated pipette tip. Transfer the remaining 750 microliters of the solution to the FACS tube labeled stained and add one microliter of HEX dye to stain the nuclei. Then mix it by slow vortexing.Load the unstained control sample into the cell sorter and record 5, 000 events. Then load the stained sample and record 5, 000 events. Draw FACS gates for identification of single nuclei.Nuclei can be selected by comparing the HEX fluorescent signal between the control and stained samples. Then sort HEX positive nuclei from the stained tube into a new 1.5 milliliter tube containing 50 microliters of PBS with 5%BSA. This protocol was used to generate single nucleus suspension directly from zebrafish brain tissue.The isolated nuclei were stained with HEX and visualized with fluorescence microscopy. They appeared intact, round, and well-separated. Importantly, nuclear aggregation was absent.Enrichment of isolated nuclei was performed with flow cytometry by gating on the presence of a HEX fluorescent signal. Unstained nuclei displayed background fluorescence while stained nuclei exhibited strong fluorescence signal. The unstained and stained nuclei were well-segregated in the violet channel.While attempting this protocol, it's important to remember that single nuclei tends to stick on a plastic material. To prevent the potential loss of nuclei and increase efficiency, basic coating of the plastic material is highly recommended. Isolated single nuclei suspension can be further utilized for single nuclei RNA This technique elucidate cellular heterogeneity in complex biological systems and help define cell subtypes and gene networks.Using a detergent and enzyme-free method registers the technical noise and bias introduced during sample preparation. This method has developed a simplified and logistical single nuclei isolation.

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