The authors wish to acknowledge support from the Hainan Provincial Natural Science Foundation of China (NO. 320QN211) and the Research Fund Program of Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture of China (NO. PBEA2021ZD01).

All animal protocols during this study were approved by the Hainan University Institutional Animal Care and Use Committee (Protocol number: HNUAUCC-2022-00063; Approval date: 2022-03-03). A list of the equipment and supplies used in this experiment can be found in the Table of Materials. A summary of the current protocol is illustrated in Figure 1.
1. Fish preparation
<ol>
	<li>Obtain 6 month old Nile tilapia with a mean body weight of 100 g from a reliable source. Select fish that are free of any signs of disease.</li>
	<li>Stop feeding the fish 24 h before proceeding to the next step. 
	NOTE: Fish fasting reduces the volume of intestinal contents and facilitates the preparation of intestinal segments.</li>
	<li>Rinse the fish in well-aerated sterile water for 15 min to wash off loosely bound bacteria on the body surface.</li>
	<li>Euthanize the fish with 300 mg/L tricaine methanesulfonate (MS-222) for about 10 min until all movement stops.</li>
</ol>
2. Reagent preparation
<ol>
	<li>Prepare 0.08% BSA-Dulbecco&#39;s phosphate-buffered saline (BSA-DPBS, for cell rinsing and resuspension). Dissolve 0.08 g of BSA (0.8% w/v) in 100 mL of 1x DPBS. Store at 4 &#176;C, and pre-cool on ice when used. 
	NOTE: The addition of BSA is beneficial for minimizing cell clumping. The BSA ratio can be increased from 0.04% to 1% according to the cell clumping conditions.</li>
	<li>Prepare the enzymatic cell dissociation reagent.
	<ol>
		<li>Dilute the collagenase/dispase with 1x PBS to a final concentration of 1 mg/mL (0.1 U/mL collagenase and 0.8 U/mL dispase). Ensure that PBS and not DPBS is used. 
		NOTE: PBS provides the calcium ions needed for the enzyme to function properly. DPBS should not be used in this step because it is Ca2+/Mg2+ free. Additionally, collagenase type I is used in the current protocol.</li>
		<li>Filter the dilution through a sterile 0.22 &#181;m membrane filter.</li>
		<li>Prepare a cell dissociation reagent that is composed of a 95% volume of collagenase/dispase solution and a 5% volume of fetal bovine serum (FBS). 
		NOTE: The dissociation reagent should be made fresh every time it is used. Using 5% FBS helps maintain the cell viability during digestion.</li>
	</ol>
	</li>
	<li>Prepare trypan blue solution.
	<ol>
		<li>Take the trypan blue solution from the 4 &#176;C refrigerator, and keep it at 15-20 &#176;C for a few minutes before use to prevent precipitation.</li>
		<li>Filter the solution through a sterile 0.22 &#181;m membrane.</li>
	</ol>
	</li>
</ol>
3. Equipment preparation
<ol>
	<li>Pre-cool the refrigerated centrifuge to 4 &#176;C before use.</li>
	<li>Obtain sterile RNase-free 1.5 mL centrifuge tubes, 50 mL conical tubes, wide-bore glass pipettes and tips, membrane filters, cell strainers, and all the low retention tubes and tips.</li>
	<li>Autoclave glass pipettes and dissection tools, including forceps, scissors, and scalpels, and pre-treat them with the RNase removal solution to prevent the adverse effects of RNase for single-cell RNA-seq.</li>
</ol>
4. Tissue dissection and cell dissociation
<ol>
	<li>Put the euthanized fish on ice for dissection. Aseptically, collect 3-4 cm of the mid-section of the intestine, and excise as much fat and mesentery as possible. Carefully remove the fat attached to the intestinal surface and the layer of elastic and malleable clear mucosa with forceps. 
	NOTE: The fat and mesentery are attached to the external surface of the intestine.</li>
	<li>Remove as much fat as possible to prevent its adverse effects on the dissociation protocol. Using a syringe, gently rinse the fragments with sterile ice-cold PBS several times to wash off the intestinal contents and mucus. Avoid rough handling.</li>
	<li>Dissect the segment of intestine into small pieces, and transfer them to a 1.5 mL tube filled with 1 mL of ice-cold PBS.</li>
	<li>Centrifuge at 300 &#215; g for 5 min at 4 &#176;C. Remove the supernatant gently, and replace with 1 mL of ice-cold 0.08% BSA-DPBS. Resuspend the tissue fragments by gentle aspiration using a glass pipette. Repeat this step twice.</li>
	<li>Remove the supernatant, and digest the tissue fragments using 1 mL of the enzymatic dissociation reagent (950 &#181;L of collagenase/dispase solution and 50 &#181;L of FBS) in a 37 &#176;C water bath for 30-60 min.
	<ol>
		<li>Additionally, add 5 &#181;L of RNase inhibitor (40 U/&#181;L in the stock solution) to the dissociation reagent if the samples are prepared for single-cell RNA sequencing. 
		NOTE: The final concentration of the collagenase/dispase mix is 1 mg/mL, including collagenase at 0.1 U/mL and dispase at 0.8 U/mL.</li>
	</ol>
	</li>
	<li>Manually invert the tube at 5 min intervals during the 30-60 min water bath. 
	NOTE: The exact incubation time varies according to the tissues used. Check the progress of dissociation under a microscope until no bulk tissue is seen. Place 10 &#181;L of the cell suspension on a glass slide with a pipette, and examine it under the microscope. Increase the digestion time if the cells are not completely dissociated.</li>
	<li>Spin down the tubes at 300 &#215; g for 5 min at 4 &#176;C, and gently remove the enzymatic cell dissociation reagent using wide-bore tips. Add 1 mL of ice-cold 0.08% BSA-DPBS, and gently pipette the cells up and down using a wide-bore glass pipette for 1 min.
	<ol>
		<li>Repeat this step twice. 
		NOTE: Pipetting up and down should be performed with care using wide-bore and low-retention tips to prevent cell disruption.</li>
	</ol>
	</li>
	<li>Pre-wet a 40 &#181;m cell strainer with 0.08% BSA-DPBS, and place it into a 50 mL conical tube. Pass the cell suspension through the strainer. Tap and rinse with an additional 200 &#181;L DPBS, and collect the suspension at the bottom of the tube.</li>
	<li>Transfer the suspension into a 1.5 mL tube. Add 5 &#181;L of DNase I (1 U/&#181;L), and incubate for 15 min at 15-20 &#176;C. Centrifuge at 300 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant, and resuspend the cell pellet in 400 &#181;L of ice-cold DPBS (Ca2+/Mg2+ free). Gently pipette up and down with wide-bore tips to mix the cells. Repeat this step twice.</li>
</ol>
5. Staining and microscopic examination
<ol>
	<li>For staining, mix the cell suspension with 0.4% trypan blue solution at a 1:1 ratio. Incubate for 3 min at room temperature.</li>
	<li>Add 5-10 &#181;L of the mixture onto a hemocytometer, and examine under the microscope. 
	NOTE: Viable cells will have unstained cytoplasm, while dead cells will have a blue cytoplasm (Figure 2).</li>
	<li>Count the viable and dead cells in three different fields under the microscope. Calculate the cell viability percentage by dividing the number of viable cells by the total number of cells in a field. 
	NOTE: The cell viability should be more than 85%, and cell concentration should not be less than 1 x 105-1 x 107/mL. The cell suspension background should be clean, without clumps, fragments, or impurities.</li>
</ol>

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<li>vanden Brink, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+sequencing+reveals+dissociation-induced+gene+expression+in+tissue+subpopulations%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations.</a> Nature Methods. 14 (10), 935-936 (2017).</li>
<li>Avey, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+RNA-seq+uncovers+a+robust+transcriptional+response+to+morphine+by+glia%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Single-cell RNA-seq uncovers a robust transcriptional response to morphine by glia.</a> Cell Reports. 24 (13), 3619-3629 (2018).</li>
<li>Herring, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unsupervised+trajectory+analysis+of+single-cell+RNA-seq+and+imaging+data+reveals+alternative+tuft+cell+origins+in+the+gut%5BTitle%5D)+AND+%22Cell+Systems%22%5BJournal%5D)">Unsupervised trajectory analysis of single-cell RNA-seq and imaging data reveals alternative tuft cell origins in the gut.</a> Cell Systems. 6 (1), 37-51 (2018).</li>
</ol>

The authors have no conflicts of interest to disclose.

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine. Before dissociation, the removal of fat and mesentery from the intestine is necessary, particularly for carnivorous fish intestines with much fat. Using a syringe instead of hard scraping to wash off the intestinal contents reduces the mechanical damage to the cells. To ensure cell viability, it is also essential to maintain the temperature at 20 &#176;C or below for the tissue dissection and rinsing steps. The was...

Cells are the fundamental units of organisms. Compared to bulk-tissue studies, single-cell level studies can reflect cell heterogeneity and provide higher-resolution information1. In recent years, researchers have applied single-cell sequencing technologies for genome, transcriptome, epigenome, or multi-omic studies at the single-cell level in mammals, zebrafish, and other model organisms and reported great breakthroughs2,3,4,5,6,7. While most studies have focused on model organisms, there are few reference protocols or commercial dissociation kits for single-cell sequencing in economic fish species, which limits the application of single-cell sequencing in aquaculture research. Therefore, developing tissue dissociation protocols that produce high-quality single-cell suspensions with high cell viability and nucleic acid integrity is crucial.
Optimizing the tissue dissociation protocol with the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. The most effective enzyme for tissue dissociation varies depending on the tissue type. In mammals, several enzymes have been used to prepare single-cell suspensions for mammalian solid tissues, including collagenase, dispase, trypsin, papain, elastase, hyaluronidase, liberase, accutase, and trypLE8,9. Trypsin digestion combined with mechanical disruption has commonly been used to dissociate tissues for cell culture in fish10,11,12,13,14. Trypsin has also been used or added into the digestion cocktail for tissue dissociation in the rat intestine15 and zebrafish gill tissue16. However, for several reasons, trypsin is not the best option for single-cell sequencing. Trypsin alone is usually ineffective for tissue dissociation. Additionally, trypsin induces DNA strand breaks17,18 and RNA degradation19.
Papain degrades the proteins that make up the tight junctions between cells. In mammalian nerve and smooth muscle cells, papain is more efficient and less destructive than other proteases20,21. However, like trypsin, papain results in the free DNA-induced aggregation of cells due to the cell lysis that occurs during enzymatic digestion9. Elastase breaks down elastin, which is typically found in the skin, lungs, ligaments, tendons, and vascular tissues22. It is often used in combination with collagenase, dispase, or trypsin for dissociating lung tissue8. Hyaluronidase cleaves the glycosidic bonds of hyaluronan, contributing to the digestion of the extracellular matrix in various connective tissues and skin9,23.
In general, collagenase and dispase are good options for extracellular matrix breakdown. They have been used in the dissociation of human, mouse, and zebrafish intestines24,25,26,27. Collagenase destroys the peptide bond in collagen, promotes the digestion of the extracellular matrix, and releases the cells into suspension, and, thus, collagenase is often used for human and mouse solid tissue dissociation, including for the liver28,29, spleen30, pancreas31, and intestine25. Dispase is a protease that hydrolyzes the N-terminal peptide bonds of nonpolar amino acid residues and is milder than collagenase. It cleaves the extracellular matrix components, such as fibronectin, type IV collagen, and to a lesser extent, type I collagen, without affecting cell-cell junctions. Dispase is used separately or with other enzymes for tissue dissociation, such as for intestine25,32, brain33, liver34, etc. In addition, commercially available digestion cocktails, including liberase, accutase, and trypLE, are also good alternatives for solid tissue dissociation, especially for the skin, liver, and kidney8,9.
Nile tilapia (Oreochromis niloticus) belongs to the family Cichlidae of the order Perciformes. It is one of the most cultured freshwater fish species in tropical and subtropical areas, with an annual production of 4.5 million tons in 202235. It is one of the best-studied aquaculture fish species with a well-annotated genome. Nile tilapia is an ideal research model for aquaculture fish species due to its short generation time, ease of rearing, and adaptability to a wide range of rearing environments. The intestine is of great research interest as it is the organ of nutrition digestion and absorption, metabolism, and mucosal immunity. The intestine is the habitat of microbial populations and is an essential immune tissue36. It is immunologically active due to the presence of numerous immune cell types, including macrophages, B cells, granulocytes, and T cells.
In the current study, we developed a protocol for preparing a high-quality single-cell suspension from the Nile tilapia intestine to facilitate single-cell level studies in aquaculture fish species. According to the characteristics of these tissue-specific enzymes and preliminary work, collagenase/dispase is appropriate for dissociating tilapia intestine tissue. The final enzyme type to consider in preparing single-cell suspensions is DNase-I, which prevents cell aggregation by degrading free DNA released through dead cell lysis during enzymatic digestion without initiating apoptotic pathways9 and increases the live cell yield36. Additionally, bovine serum albumin (BSA) is added to the washing buffer to reduce cell clumping and improve cell viability. Several reagent companies describe BSA as an enzyme stabilizer. The addition of 0.04%-1% BSA to PBS (phosphate-buffered saline) has been used to develop a washing solution for preparing single-cell sequencing suspensions without adverse effects38. The addition of a low ratio of BSA could help maintain cell viability and avoid the free DNA-induced aggregation of cells due to cell lysis. This protocol can also provide a valuable reference for developing cell dissociation protocols from the intestines of other aquaculture fish species.

<table><tbody><tr><td>0.22-&amp;mu;m Sterile Filter</td><td>Solarbio Life Sciences</td><td>SLGV033RB</td><td>It is used to filter and sterilize the enzyme solution.</td></tr><tr><td>40-&amp;mu;m Cell Strainer</td><td>Solarbio Life Sciences</td><td>F8200</td><td>Cell Strainer is applied to eliminate undigested tissue pieces.</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>SRE0098</td><td>Powder; dilute 0.04 g BSA with 100 mL 1&amp;times; DPBS to prepare 0.04% BSA-DPBS washing bffer. Store at 2 - 8 &amp;deg;C.</td></tr><tr><td>Collagenase II</td><td>Sangon Biotech</td><td>A004202</td><td>Dilute with PBS to a final concentration of 1 mg/mL.</td></tr><tr><td>Collagenase/dispase</td><td>Roche</td><td>10269638-001</td><td>Dilute with PBS to a final concentration of 1 mg/mL.</td></tr><tr><td>Dispase</td><td>Sigma-Aldrich</td><td>D4818</td><td>Dilute with PBS to a final concentration of 1 mg/mL.</td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>AMPD1</td><td>DNase I helps reduce cell clumping.</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS), Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;-free</td><td>Solarbio Life Sciences</td><td>E607009-0500</td><td>Store at room temperature.</td></tr><tr><td>Elastase</td><td>Sangon Biotech</td><td>A600438</td><td>Dilute with PBS to a final concentration of 0.5 mg/mL.</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>Serum, used at volume of 5% in digetstion solution.</td></tr><tr><td>Inverted&amp;nbsp;Microscope</td><td>Leica</td><td>qTOWER3G</td><td>It is used to examine cell viability.</td></tr><tr><td>Liberase</td><td>Roche</td><td>5401119001</td><td>Dilute with PBS to a final concentration of 0.25 mg/mL.</td></tr><tr><td>Nile tilpia (&lt;em&gt;Oreochromis niloticus&lt;/em&gt;)</td><td>ProGift Aquaculture Technology Co. Ltd.</td><td>NA</td><td>Healthy fish with no disease signs (Mean body weight: 100 g).&amp;nbsp;</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Solarbio Life Sciences</td><td>P1020</td><td>Store at room temperature.</td></tr><tr><td>Refrigerated Centrifuge</td><td>Eppendorf</td><td>5424</td><td>It is used to spin down the tissue and cell petet.</td></tr><tr><td>RNase inhibitor</td><td>NEB</td><td>M0314L</td><td>Inhibit RNase activity</td></tr><tr><td>Solid-phase RNase-Be-Gone Reagent</td><td>Sangon Biotech</td><td>B644201-0050</td><td>It is used to remove the RNase from tools such as dissecting scissors and glass pipettes. Store at room temperature.</td></tr><tr><td>Tricaine methanesulfonate (MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td>For fish euthanasia.&amp;nbsp;</td></tr><tr><td>Trypan Blue</td><td>Invitrogen</td><td>C0040</td><td>It is used for staining dead cells.</td></tr><tr><td>Trypsin</td><td>Sangon Biotech</td><td>E607001</td><td>Dilute with PBS to a final concentration of 1 mg/mL.</td></tr></tbody></table>

single-cell suspension preparation from nile tilapia intestine for single-cell sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine for single-cell sequencing (Figure 1). This research shows that the collagenase/dispase mix has a good dissociation effect and is mild for intestine tissue. The selection of the optimal digestion enzyme is essential for preparing a high-quality single-cell suspension. In the preliminary work, the dissociation efficiencies of several commonly used enzymes were compared, and the results a...

Watch this Scientific Journal Video about Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing at JoVE.com

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

1.7K Views.  Hainan University. Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Video: Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Environment

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

Developmental Biology

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

Immunology and Infection

Gentle Isolation of Nuclei from the Brain Tissue of Adult African Turquoise Killifish, a Naturally Short-Lived Model for Aging Research

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we demonstrate a protocol to generate single-nucleus RNA sequencing (snRNA-seq) libraries from the brains of the naturally short-lived vertebrate African turquoise killifish Nothobranchius furzeri. The African turquoise killifish has a lifespan of 4-6 months and can be housed in a cost-effective manner, thus reducing cost and time barriers to study vertebrate brain aging. However, tailored protocols are needed to isolate nuclei of sufficient quality for downstream single-cell experiments from the brain of young and aged fish. Here, we demonstrate an empirically optimized protocol for the isolation of high-quality nuclei from the brain of adult African turquoise killifish, a critical step in the generation of high-quality single nuclei omic libraries. Furthermore, we show that the steps to reduce contaminating background RNA are important to clearly distinguish cell types. In summary, this protocol demonstrates the feasibility of studying brain aging in non-traditional vertebrate model organisms.

Here we present a protocol to isolate nuclei from the brains of the short-lived vertebrate model Nothobranchius furzeri for downstream applications such as single-nucleus RNA sequencing or single-nucleus assay for transposase-accessible chromatin with sequencing (ATAC-seq).

Studying brain aging at single-cell resolution in vertebrate systems remains challenging due to cost, time, and technical constraints. Here, we ...

Neuroscience

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 ...

Isolation of Endothelial Cells from the Lumen of Mouse Carotid Arteries for Single-Cell Multi-Omics Experiments

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in the endothelium at the transcriptomic and epigenomic levels, resulting in proatherogenic responses. Recently, single-cell RNA sequencing (scRNAseq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATACseq) studies were performed to determine the transcriptomic and chromatin accessibility changes at a single-cell resolution using the mouse partial carotid ligation (PCL) model. As endothelial cells (ECs) represent a minor fraction of the total cell populations in the artery wall, a luminal digestion method was used to obtain EC-enriched single-cell preparations. For this study, mice were subjected to PCL surgery to induce d-flow in the left carotid artery (LCA) while using the right carotid artery (RCA) as a control. The carotid arteries were dissected out two days or two weeks post PCL surgery. The lumen of each carotid was subjected to collagenase digestion, and endothelial-enriched single cells or single nuclei were obtained. These single-cell and single-nuclei preparations were subsequently barcoded using a 10x Genomics microfluidic setup. The barcoded single-cells and single-nuclei were then utilized for RNA preparation, library generation, and sequencing on a high-throughput DNA sequencer. Post bioinformatics processing, the scRNAseq and scATACseq datasets identified various cell types from the luminal digestion, primarily consisting of ECs. Smooth muscle cells, fibroblasts, and immune cells were also present. This EC-enrichment method aided in understanding the effect of blood flow on the endothelium, which could have been difficult with the total artery digestion method. The EC-enriched single-cell preparation method can be used to perform single-cell omics studies in EC-knockouts and transgenic mice where the effect of blood flow on these genes has not been studied. Importantly, this technique can be adapted to isolate EC-enriched single cells from human artery explants to perform similar mechanistic studies.

We present a method for isolating endothelial cells and nuclei from the lumen of mouse carotid arteries exposed to stable or disturbed flow conditions to perform single-cell omics experiments.

Atherosclerosis is an inflammatory disease of the arterial regions exposed to disturbed blood flow (d-flow). D-flow regulates the expression of genes in ...

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 ...

Two-Step Cell Digestion of Mouse Carotid Artery

Preparation of a Single-Cell Suspension from Mouse Carotid Arteries for Single-Cell Sequencing

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are clogged by plaque. Revealing the cellular composition of the carotid artery at the single-cell level is essential for treating carotid atherosclerosis. However, there is no ready-to-use protocol for the preparation of single-cell suspensions from carotid arteries. To obtain a suitable protocol for the dissociation of normal carotid arteries at the single-cell level with less damage to cells, we designed a two-step digestion method by integrating the digestion process of collagenase/DNase and trypsin. Acridine orange/propidium iodide (AO/PI) dual-fluorescence counting was used to detect cell viability and concentration, and it was found that the single-cell suspension satisfied the requirements for single-cell sequencing, with the viability of cells over 85% and a high cell concentration. After single-cell data processing, a median of ~2500 transcripts per cell were detected in each carotid artery cell. Notably, a variety of cell types of the normal carotid artery, including vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages and dendritic cells (M&#966;/DCs), were concurrently detectable. This protocol may be applied to prepare a single-cell suspension of blood vessels from other tissues with appropriate modifications.

Author Spotlight: Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid arteries.

Carotid arteries are major blood vessels in the neck that supply blood and oxygen to the brain, but carotid stenosis occurs when carotid arteries are ...

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Summary

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