This work was funded by the friends of Sophia Foundation (SSWO WAR-63).

All zebrafish husbandry and experiments were conducted according to the institutional guidelines of the Erasmus MC and Animal welfare legislation. The use of zebrafish larvae at 5 days post fertilization falls under the category of experiments that do not require formal ethical approval, as outlined by the Dutch regulations.
1. Obtaining 5 days post fertilization (dpf) wildtype and tg(phox2bb:GFP) larvae
<ol>
	<li>Set up breeding of wildtype zebrafish and collect 50 eggs in HEPES-buffered E3 medium (hereafter referred to as E3) in a 15 cm Petri dish. Use these fish as a negative control for fluorescence-activated cell sorting (FACs).</li>
	<li>Set up breeding of tg(phox2bb:GFP) zebrafish and collect approximately 300 eggs in E3 in a 15 cm Petri dish.</li>
	<li>Keep fertilized eggs in E3 (maximum 50 eggs/dish) on a 14 h/10 h light/dark cycle, in an incubator at 28.5 &#176;C.</li>
	<li>At 1 dpf, remove unfertilized eggs and let the fertilized ones develop to 5 dpf.</li>
	<li>Select tg(phox2bb:GFP) larvae at 1 dpf.</li>
</ol>
2. Wildtype whole larvae dissociation
<ol>
	<li>Anesthetize 5 dpf larvae with 0.016% Tricaine.</li>
	<li>Chop 30 zebrafish into small pieces using a razor blade in a Petri dish containing 1 mL of 10x trypsin-EDTA solution.</li>
	<li>Transfer the chopped zebrafish into a microcentrifuge tube with a total volume of 2 mL of 10x trypsin-EDTA solution and leave on ice for 3 h. Pipette up and down with a P1000 pipette every hour to stimulate dissociation.</li>
</ol>
3. Gut isolation
<ol>
	<li>Place 6-10 5 dpf larvae anesthetized with 0.016% Tricaine in a row, on a 1.8% agarose plate (0.45 g of agarose in 25 mL of E3), under a dissection microscope</li>
	<li>Put one insect pin in the head of the zebrafish.</li>
	<li>Remove all remaining E3 using a tissue.</li>
	<li>Isolate the intestine using another insect pin, without disturbing any other organs. Make sure that the yolk is removed (Supplementary Figure S1A).</li>
	<li>Once isolated, inspect the intestine and remove all non-intestinal material (e.g., skin, fat, liver) if needed.</li>
	<li>Collect the intestine with tweezers and place it in a microcentrifuge tube, containing phosphate-buffered saline (PBS) with 10% fetal calf serum (FCS), on ice. 
	&#8203;NOTE: A minimum of 244 intestines should be isolated, keeping the total dissection time at 3 h. This is feasible with two people working in parallel.</li>
</ol>
4. Gut dissociation
<ol>
	<li>Immediately after the dissection of all intestines, centrifuge the microcentrifuge tube at full speed (13,800 &#215; g) for 30 s.</li>
	<li>Remove the PBS/10% FCS but leave a small amount (~100 &#181;L) to prevent the intestines from drying out. Add 500 &#181;L of 2.17 mg/mL papain in HBSS containing CaCl2 and MgCl2, to dissociate cells.</li>
	<li>Activate papain with 2.5 &#181;L of cysteine (1 M).</li>
	<li>Incubate the guts in a water bath at 37 &#176;C for 10 min. Pipette up and down halfway (after 5 min) to stimulate enzymatic tissue digestion.</li>
	<li>Transfer cells into a FACS tube using a 35 &#181;m cell strainer, prewetted with 0.5 mL of PBS/10% FCS.</li>
	<li>Wash the strainer by adding a total of 2 mL of PBS/10% FCS in 0.5 mL steps.</li>
	<li>Centrifuge at 700 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the pellet in 300 &#181;L of PBS/10% FCS.</li>
	<li>Add 1 &#181;g/mL of 4&#39;,6-diamidino-2-phenylindole (DAPI) to label dead cells (1:1,000) and incubate for 5 min to allow their exclusion during FACS.</li>
</ol>
5. FACS enrichment
<ol>
	<li>Use the wildtype sample to set the gates of the sorted cell population by recording 3,000 cells on the flow cytometer (Supplementary Figure S1B-E).</li>
	<li>Use a 100 &#181;m nozzle, set sort precision on purity, and put the collection tube containing 200 &#181;L of PBS/5% FCS, in the FACS machine.</li>
	<li>Load the cell suspension of the guts and sort live (DAPI-negative), single cells into the collection tube (Figure 2C), by excluding doublets (Figure 2B) and dead cells (Figure 2C). 
	NOTE: For the tg(phox2bb) zebrafish, the proportion of GFP+ cells is only checked for reference, as all single live cells are sorted.</li>
	<li>Keep the collection tube on ice after cell sorting.</li>
	<li>Count the cell suspension with a hemocytometer, including a viability check with trypan blue. If the viability is at least 80%, continue with the next step.</li>
	<li>Cells are now ready to process for scRNAseq (i.e., 10x Genomics Chromium platform). Use approximately 20,000 cells per sample.</li>
</ol>

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

<ol>
	<li>Saldana-Morales, F. B., Kim, D. V., Tsai, M. T., Diehl, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healthy+intestinal+function+relies+on+coordinated+enteric+nervous+system,+immune+system,+and+epithelium+eesponses.">Healthy intestinal function relies on coordinated enteric nervous system, immune system, and epithelium eesponses.</a> Gut Microbes. 13 (1), 1-14 (2021).</li><li>Sitrin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Gastrointestinal System. , (2014).</li><li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organisation+of+the+autonomic+nervous+system:+peripheral+connections.">The organisation of the autonomic nervous system: peripheral connections.</a> Autonomic Neuroscience: Basic and Clinical. 130 (1-2), 1-5 (2006).</li><li>Furness, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enteric+nervous+system+and+neurogastroenterology.">The enteric nervous system and neurogastroenterology.</a> Nature Reviews. Gastroenterology &amp; Hepatology. 9 (5), 286-294 (2012).</li><li>Obata, Y., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+microbiota+and+the+immune+system+on+the+development+and+organization+of+the+enteric+nervous+system.">The effect of microbiota and the immune system on the development and organization of the enteric nervous system.</a> Gastroenterology. 151 (5), 836-844 (2016).</li><li>Heuckeroth, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hirschsprung+disease+-+integrating+basic+science+and+clinical+medicine+to+improve+outcomes.">Hirschsprung disease - integrating basic science and clinical medicine to improve outcomes.</a> Nature Reviews. Gastroenterology &amp; Hepatology. 15 (3), 152-167 (2018).</li><li>Antonucci, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+intestinal+pseudo-obstruction.">Chronic intestinal pseudo-obstruction.</a> World Journal of Gastroenterology. 14 (19), 2953-2961 (2008).</li><li>De Giorgio, R., Sarnelli, G., Corinaldesi, R., Stanghellini, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+our+understanding+of+the+pathology+of+chronic+intestinal+pseudo-obstruction.">Advances in our understanding of the pathology of chronic intestinal pseudo-obstruction.</a> Gut. 53 (11), 1549-1552 (2004).</li><li>Bianco, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+neuromyopathies:+highlights+on+genetic+mechanisms+underlying+chronic+intestinal+pseudo-obstruction.">Enteric neuromyopathies: highlights on genetic mechanisms underlying chronic intestinal pseudo-obstruction.</a> Biomolecules. 12 (12), 1849 (2022).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nature Reviews. Genetics. 8 (5), 353-367 (2007).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+growth+and+differentiation+in+zebrafish.">Intestinal growth and differentiation in zebrafish.</a> Mechanisms of Development. 122 (2), 157-173 (2005).</li><li>Wallace, K. N., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+and+conserved+aspects+of+gut+development+in+zebrafish.">Unique and conserved aspects of gut development in zebrafish.</a> Developmental Biology. 255 (1), 12-29 (2003).</li><li>Harrison, C., Wabbersen, T., Shepherd, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+the+development+of+the+enteric+nervous+system+using+a+Tg(-8.3bphox2b:Kaede)+transgenic+zebrafish.">In vivo visualization of the development of the enteric nervous system using a Tg(-8.3bphox2b:Kaede) transgenic zebrafish.</a> Genesis. 52 (12), 985-990 (2014).</li><li>Kuil, L. E., Chauhan, R. K., Cheng, W. W., Hofstra, R. M. W., Alves, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+organism+for+studying+enteric+nervous+system+development+and+disease.">Zebrafish: a model organism for studying enteric nervous system development and disease.</a> Frontiers in Cell and Developmental Biology. 8, 629073 (2020).</li><li>Stuart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Integration+of+Single-Cell+Data.">Comprehensive Integration of Single-Cell Data.</a> Cell. 177 (7), 1888-1902 (2019).</li><li>Kuil, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+characterization+of+the+larval+zebrafish+enteric+nervous+system+at+a+single+cell+transcriptomic+level.">Unbiased characterization of the larval zebrafish enteric nervous system at a single cell transcriptomic level.</a> iScience. 26 (7), 107070 (2023).</li><li>Gao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+differential+transcriptomes+and+cell+types+in+zebrafish+larvae+intestine+and+liver.">Unraveling differential transcriptomes and cell types in zebrafish larvae intestine and liver.</a> Cells. 11 (20), 3290 (2022).</li><li>Jin, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdx1b+protects+intestinal+cell+fate+by+repressing+signaling+networks+for+liver+specification.">Cdx1b protects intestinal cell fate by repressing signaling networks for liver specification.</a> Journal of Genetics and Genomics. 49 (12), 1101-1113 (2022).</li><li>Willms, R. J., Jones, L. O., Hocking, J. C., Foley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+atlas+of+microbe-responsive+processes+in+the+zebrafish+intestine.">A cell atlas of microbe-responsive processes in the zebrafish intestine.</a> Cell Reports. 38 (5), 110311 (2022).</li><li>Kline, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fishing for answers: Isolating enteric neurons and identifying putative ENS mutants. , (2016).</li><li>Allan, K., DiCicco, R., Ramos, M., Asosingh, K., Yuan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+a+single+cell+suspension+from+zebrafish+retinal+tissue+for+flow+cytometric+cell+sorting+of+Muller+glia.">Preparing a single cell suspension from zebrafish retinal tissue for flow cytometric cell sorting of Muller glia.</a> Cytometry A. 97 (6), 638-646 (2020).</li><li>Lopez-Ramirez, M. A., Calvo, C. F., Ristori, E., Thomas, J. L., Nicoli, 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+and+culture+of+adult+zebrafish+brain-derived+neurospheres.">Isolation and culture of adult zebrafish brain-derived neurospheres.</a> Journal of Visualized Experiments. 53617 (108), 53617 (2016).</li></ol>

The authors have no conflicts of interest to disclose.

Here, we present a method for isolation and dissociation of the gut of 5 dpf zebrafish larvae using FACS. With this method, different intestinal cell types were successfully collected and analyzed by scRNA-seq, using the 10x Genomics Chromium platform. We selected the tg(phox2bb:GFP) zebrafish line, as we wanted an indication that viable ENS cells would be also isolated (Figure 2D). However, it is important to note that this method can be readily extended to other zebrafish lines of...

The gastrointestinal (GI) tract is a complex system that plays a vital role in overall health and well-being. It is responsible for the digestion and absorption of nutrients, as well as the elimination of waste products1,2. The GI tract is composed of multiple cell types, including epithelial cells, smooth muscle cells, immune cells, and the enteric nervous system (ENS), which communicate closely together to regulate and maintain proper intestinal function3,4,5. Defects in the development of the GI tract can have far-reaching effects on various aspects such as nutrient absorption, microbiota composition, the gut-brain axis, and the ENS, leading to several enteric neuromuscular disorders, such as Hirschsprung disease and Chronic intestinal pseudo-obstruction6,7. These disorders are characterized by severe gut dysmotility caused by alterations in various key cells, such as the interstitial cells of Cajal, smooth muscle cells, and the ENS6,8,9. However, the molecular mechanisms underlying GI development and dysfunction are still poorly understood.
The zebrafish is a valuable model organism for studying GI development and dysfunction due to its rapid embryonic development, transparency during embryonic and larval stages, and genetic tractability10,11,12,13,14. Numerous transgenic zebrafish lines expressing fluorescent proteins are available. An example of such a line is the tg(phox2bb:GFP) zebrafish, commonly used to study the ENS, as all phox2bb+ cells, including enteric neurons, are labeled15,16. Here, using the tg(phox2bb:GFP) zebrafish line, we present a method for intestinal isolation of 5 days post fertilization (dpf) larvae for single-cell RNA sequencing (scRNA-seq) analysis (Figure 1).

<table><tbody><tr><td>10x Trypsin (0.5%)-EDTA (0.2%)</td><td>Sigma</td><td>59418C</td><td></td></tr><tr><td>5 mL round bottom tube with cell-strainer cap</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>BD Falcon Round-Bottom Tube 5 mL (FACS tubes) snap cap</td><td>BD Biosciences</td><td>352054</td><td></td></tr><tr><td>Cell Ranger v3.0.2</td><td>10X Genomics</td><td>N/A</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>Cat#D-9542</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus SZX16</td><td></td><td></td></tr><tr><td>FACSAria III sorter machine</td><td>BD Biosciences</td><td>N/A</td><td></td></tr><tr><td>HBSS with CaCl&lt;sub&gt;2 &lt;/sub&gt;and MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Gibco</td><td>14025050</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>26000-25</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>C7352</td><td></td></tr><tr><td>MS-222, Tricaine</td><td>Supelco</td><td>A5040-250G</td><td></td></tr><tr><td>Papain</td><td>Sigma</td><td>P4762</td><td></td></tr><tr><td>Seurat v3</td><td>Stuart et&amp;nbsp;al. (2019)</td><td>N/A</td><td></td></tr><tr><td>Trypan blue&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>Cat#T8154</td><td></td></tr></tbody></table>

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.

With this protocol, we achieved successful isolation and dissociation of entire intestines from 5 dpf&#160;larvae. Using papain as the dissociation enzyme, we significantly enhanced cell viability, enabling the capture of 46,139 events involving single, viable cells (6.4% of all cells) out of 244 isolated guts (Figure 2A). Wildtype whole larvae were used as a control to ensure that the sorting process was optimized, enabling effective cell identification and sorting. Whole larvae could be us...

Watch this Scientific Journal Video about Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development at JoVE.com

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.

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 ...

gut-isolation-from-zebrafish-larvae-for-single-cell-rna-sequencing

Research

JoVE Journal

Developmental Biology

1.5K Views.  Erasmus University Medical Center - Sophia Children's Hospital. Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

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

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Genetics

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Biology

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Immunology and Infection

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

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 ...

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.

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 ...