The authors appreciate the advice and technical support provided by Dr. Richard Konz and staff at the Flow Cytometry Core facility at the University of Massachusetts Medical School. Core resources supported by the Diabetes Endocrinology Research Center Grant DK032520 were also used. This work is supported by grants from the National Institute of Mental Health (NIMH), the National Institute of Drug Abuse (NIDA) and the National Institute of Child Health and Human Development.

Nuclei Extraction
<ol>
<li>Put&nbsp;250 mg of frozen postmortem brain tissue in 5 mL Lysis Buffer1 in a douncer and place it on ice. To receive about 5 million nuclei after FACS sorting, we usually use 1000 mg of tissue per sample.</li>
<li>Once the tissue has thawed, dounce the tissue for 1 minute while on ice.</li>
<li>Take the homogenized tissue and place it into a 15 mL clear ultracentrifuge tube. Put the tube on ice.</li>
<li>Take 9 mL of Sucrose Solution2 with a pipette, put the pipette at the bottom of the clear ultracentrifuge tube, and release the Sucrose Solution2 to create a concentration gradient with the homogenized tissue solution on top of the Sucrose Solution2.</li>
<li>Weigh the ultracentrifuge tubes to make sure that they will all be balanced while rotating within the ultracentrifuge. Make any adjustments to the weight by adding Lysis Buffer1 to the top layer.</li>
<li>After the tubes have been weighed, place them into the rotor's buckets.</li>
<li>Place all buckets into a SW28 rotor and carefully place the rotor into the ultracentrifuge.</li>
<li>Ultracentrifuge the samples at 107163.6 RCF for 2.5 hours at 4&deg;C.</li>
<li>Once the centrifugation is complete, remove the supernatant, along with the layer of debris found at the concentration gradient, with the use of a vacuum, while being careful not to disturb the pellet containing the nuclei at the bottom of the tube.</li>
<li>Add 500 &micro;L of 1X PBS to each pellet and let sit on ice for 20 minutes. This allows the pellet to dissolve a little on its own, making it easier to later mechanically dissolve it by pipetting up and down, which lessens the amount of stress the nuclei experience.</li>
<li>After the nuclei have incubated on ice for 20 minutes, mechanically pipette up and down to completely dissolve the nuclei within 1XPBS.</li>
<li>Remove the nuclei-containing solution from the ultracentrifuge tubes and combine the contents of two tubes into a single 2 mL microcentrifuge tube. Again, place the samples on ice.</li>
</ol>
Immunostaining for FACS
<ol>
<li>At this point, make an immunostaining mixture composed of primary and secondary antibody, and Blocking Mix. For each sample, combine 300 &micro;L of 1XPBS containing 1.2 &micro;L of NeuN antibody, 100 &micro;L of Blocking Mix, which contains 0.5% BSA and 10% Normal Goat Serum in 1XPBS, and 1 &micro;L of Alexa Fluor 488. For the control samples, exclude the primary antibody NeuN and, instead, just add more 1XPBS.</li>
<li>Vortex the immunostaining mixture and incubate for 5 minutes at room temperature in the dark.</li>
<li>While the staining mixture is incubating, make the controls for the samples. For FACS sorting, a sample, containing nuclei alone, called the "unstained control" is needed; a second sample, a negative control, containing the nuclei with the secondary antibody Alexa Fluor 488 without the primary antibody NeuN is also needed.</li>
<li>Aliquot 20 &micro;L of the nuclei containing solution into a 2 mL microcentrifuge tube containing 1XPBS for the unstained control, and another 20 &micro;L into another 2 mL tube containing 980 uL of 1XPBS for the negative control. Raise the volume of the nuclei-containing sample back up to 1 mL with 1XPBS. All samples are placed back on ice.</li>
<li>Now that the immunostaining mixture has finished incubating, add its contents of 401 &micro;L into the appropriate samples. The nuclei sample will receive the mixture containing both NeuN and Alexa Fluor 488, while the negative sample&nbsp;will receive the mixture containing only the secondary antibody Alexa Fluor 488.</li>
<li>Take the samples into the cold room to rotate in the dark for 45 minutes.</li>
<li>After the samples have incubated in the dark for 45 minutes, put them on ice and bring them to the FACS facility. During the transport to the FACS facility, the samples should be kept on ice and in the dark.</li>
</ol>
FACS
<ol>
<li>At the FACS facility, filter the samples through a 40 um filter. Then, run&nbsp;them&nbsp;through the instrument for a few minutes, while being kept cold, in order to get some preliminary data before sorting.</li>
<li>At this point, it is necessary to set the appropriate gates required for sorting. Several different gates are used to sort samples. The first gate gives an idea of the size of the different nuclei found within the sample, and&nbsp;allows separatation of&nbsp;debris from actual nuclei. The second gate allows us to sort nuclei individually. Discard any aggregates are discarded at this point. Finally, the third gate is set to a specific fluorescence wavelength, matching that of the fluorochrome found in our secondary antibody. Placing this gate allows us to sort and separate NeuN +, hence neuronal nuclei from NeuN - nuclei.</li>
<li>After all the gates are chosen, the nuclei can be sorted into 1XPBS.</li>
<li>Once the whole sample has gone through, check the purity of the sort by running an aliquot of the sorted sample through the instrument. Make sure that the sample's fluorescence is not spread out, but rather is contained within a single quadrant. It is best to choose samples which are over 90% pure.</li>
</ol>
After FACS
<ol>
<li>Once the sample has been sorted, processing of the nuclei can begin. Take 10 mL of sorted sample and add to it 2 mL of Sucrose Solution2, 50 uL of 1M CaCl2 and 30 ul of 1m Mg(Ace)2. As a reminder, always be sure to keep the sample on ice since the nuclei are unfixed.</li>
<li>If there is less than 10 mL of sorted sample, raise the volume up to 10 mL by adding 1XPBS, or adjust the additional materials accordingly.</li>
<li>Invert the sample several times and place on ice for 15 minutes.</li>
<li>After the samples have incubated on ice for 15 minutes, centrifuge them in a swing bucket rotor at 1786 RCF for 15 minutes at 4&deg;C.</li>
<li>Discard the supernatant with the use of a vacuum, without disturbing the white pellet found at the bottom of the tube.</li>
<li>Dissolve the pellet in 200 &micro;l of whatever solution you require for the proceeding experiments, and pipette up and down.</li>
<li>Transfer the solution into a smaller tube and place it into the -80&deg;C freezer until further use.</li>
</ol>

<ol>
	<li>Siegmund, K. D., Connor, C. M., Campan, M., Long, T. I., Weisenberger, D. J., Biniszkiewicz, D., Jaenisch, R., Laird, P. W., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+the+human+cerebral+cortex+is+dynamically+regulated+throughout+the+life+span+and+involves+differentiated+neurons.">DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.</a> PLoS ONE. 2, 895-895 (2007).</li><li>Jiang, Y., Matevossian, A., Huang, H. S., Straubhaar, J., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Spalding, K. L., Bhardwaj, R. D., Buchholz, B. A., Druid, H., Frisen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Acevedo, L. G., Iniguez, A. L., Holster, H. L., Zhang, X., Green, R., Farnham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+ChIP-chip+analysis+using+10,000+human+cells.">Genome-scale ChIP-chip analysis using 10,000 human cells.</a> Biotechniques. 43, 791-797 (2007).</li></ol>

The protocol presented here should be particularly useful for studies focused on epigenetic changes of neuronal chromatin and, more generally, on the molecular phenotype of the neuronal nucleus, during normal developing and aging, or in neurological or psychiatric disease. The method is of particular advantage when the cellular heterogeneity of brain tissue, including potential shifts in neuron-to-glia ration during the course of aging or due to disease are a concern 1. For example, by combining the present so...

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			<div>
			Reagents:

<table>
<tr><th colspan="3" align="left">1. Lysis Buffer</th></tr>
<tr><td>0.32M</td><td>Sucrose</td><td>5.47 g</td></tr>
<tr><td>5 mM</td><td>CaCl2</td><td>250 &micro;l</td></tr>
<tr><td>3 mM</td><td>Mg(Acetate)2</td><td>150 &micro;l</td></tr>
<tr><td>0.1 mM</td><td>EDTA</td><td>10 &micro;l</td></tr>
<tr><td>10mM</td><td>Tris-HCl, pH8</td><td> 500 &micro;l</td></tr>
<tr><td>1 mM </td><td>DTT</td><td>17 &micro;l</td></tr>
<tr><td>0.1%</td><td>Triton X-100</td><td>50 &micro;l</td></tr>
<tr><td colspan="3" align="center" style="padding:8px 0 16px 0">Adjust volume to 50 ml with Autoclaved water</td></tr>
</table>
<table>
<tr><th colspan="3" align="left">2. Sucrose Solution</th></tr>
<tr><td>1.8 M</td><td>Sucrose</td><td>30.78 g</td></tr>
<tr><td>3 mM </td><td>Mg(Acetate)2</td><td>150 &micro;l</td></tr>
<tr><td>1 mM</td><td>DTT</td><td>17 &micro;l</td></tr>
<tr><td>10 mM</td><td>Tris-HCl, pH8</td><td>500 &micro;l</td></tr>
<tr><td colspan="3" align="center" style="padding:8px 0 16px 0">Adjust volume to 50 ml with Autoclaved water</td></tr>
</table>
<table>
<tr><th colspan="3" align="left">3. Dounce buffer</th></tr>
<tr><td>10 mM</td><td>Tris</td><td>0.242 g</td></tr>
<tr><td>4 mM</td><td>MgCl2</td><td>0.163 g</td></tr>
<tr><td>1 mM</td><td>CaCl2</td><td>0.03 g</td></tr>
<tr><td colspan="3" align="center" style="padding:8px 0 16px 0">Adjust pH to 7.5 and volume to 200 ml with Autoclaved water</td></tr>
</table>		</div>

neuronal nuclei isolation from human postmortem brain tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

Watch this Scientific Journal Video about Neuronal Nuclei Isolation from Human Postmortem Brain Tissue at JoVE.com

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

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22.0K Views.  University of Massachusetts Medical School. The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain. 

Video: Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

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.

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 ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy homeostasis and the metabolism of xenobiotics, among others. These tasks are performed through tight coordination between hepatic parenchymal and non-parenchymal cells. Additionally, various metabolic activities are confined to specific areas of the hepatic lobule-a phenomenon called liver zonation. Recent advances in single-cell sequencing technologies have empowered researchers to investigate tissue heterogeneity at a single-cell resolution. In many complex tissues, including the liver, harsh enzymatic and/or mechanical dissociation protocols can negatively affect the viability or the quality of the single-cell suspensions needed to comprehensively characterize this organ in health and disease.
This paper describes a robust and reproducible protocol for isolating nuclei from frozen, archived liver tissues. This method yields high-quality nuclei that are compatible with downstream, single-cell omics approaches, including single-nucleus RNA-seq, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), as well as multimodal omics (joint RNA-seq and ATAC-seq). This method has been successfully used for the isolation of nuclei from healthy and diseased human, mouse, and non-human primate frozen liver samples. This approach allows the unbiased isolation of all the major cell types in the liver and, therefore, offers a robust methodology for studying the liver at the single-cell resolution.

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

The liver is a complex and heterogenous tissue responsible for carrying out many critical physiological functions, such as the maintenance of energy ...

Isolation and Profiling of Single Nuclei from Frozen Mammalian Tissues

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

Author Spotlight: Enhancing Drug Discovery - Development of Automated, Standardized Protocols for Nuclei Extraction from Frozen Tissues 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.