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, ...

neuronal-nuclei-isolation-from-human-postmortem-brain-tissue

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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 ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

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 ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

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.