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 &amp;mu;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 &amp;mu;L, 200 &amp;mu;L, 1000 &amp;mu;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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 ...

nuclei-isolation-from-whole-tissue-using-detergent-enzyme-free

Nanyang Technological University

Research

JoVE Journal

Biology

24.5K Views.  Université Libre de Bruxelles (ULB). Het voorbereiden van een enkele cel of enkele kernen schorsing is een cruciale stap voor hoge doorvoer transcriptome en epigenoom profilering. Ons protocol biedt een eenvoudige, reproduceerbare en universele methode voor enkele kernisolatie. In tegenstelling tot andere methoden die wasmiddel voor kernisolatie, ons protocol is kolom-gebaseerd, dat is wasmiddel en enzym-vrij.Het maakt kernisolatie in minder dan 30 minuten mogelijk. Ons protocol vereenvoudigt de isolatie van kernen. Het b...

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

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Medicine

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

Genetics

Isolation of Nuclei for Multiome Sequencing in Solid Tumor Specimen

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Author Spotlight: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

Cancer Research

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Developmental Biology

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Neuroscience

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 ...

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 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.

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

Isolation of Nuclei from Fresh Frozen Glioma Tissues: A Method to Obtain Intact Nuclei from Glioma Tumor Samples

Source: Narayanan, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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)
This video describes the protocol to isolate single-nuclei from fresh frozen glioma tissue. The isolated single-nuclei can be used as specimens for sequencing techniques such as single-nuclei RNA (snRNA) sequencing and single-nuclei Assay for Transposase-Accessible Chromatin (snATAC) sequencing to obtain deeper insights into inter- and intra-tumor heterogeneity observed in glioma of the central nervous tissues.

Source: Narayanan, A. et al. Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq. J. Vis. Exp. (2020)
This video ...

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

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