Name: multiplexed single cell mrna sequencing analysis of mouse embryonic cells
Uploaded: 2020-01-07T14:00:00
Description: Watch this Scientific Journal Video about Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells at JoVE.com

We thank David M. Patterson and Christopher S. McGinnis from Dr. Zev J. Gartner lab for their kind supply of the lipid based barcoding reagents and suggestions on the experimental steps and data analysis. This work was founded by the National Institutes of Health (HL13347202).

The animal procedure is in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC).
1. Mouse Embryonic Heart Dissection and Single Cell Suspension Preparation
NOTE: This step could take a few hours depending on the numbers of embryos to dissect.
<ol>
	<li>To acquire E18.5 embryonic hearts, euthanize a pregnant CD1 mouse by CO2 administration. Use a razor to remove the unwanted hair in the abdominal area and disin...

<ol>
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M., 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=Single-cell+resolution+of+temporal+gene+expression+during+heart+development.">Single-cell resolution of temporal gene expression during heart development.</a> Developmental Cell. 39 (4), 480-490 (2016).</li><li>Li, G., 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=Single+cell+expression+analysis+reveals+anatomical+and+cell+cycle-dependent+transcriptional+shifts+during+heart+development.">Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development.</a> Development. 146 (12), dev173476 (2019).</li><li>Meilhac, S. M., Buckingham, M. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genome+sequencing:+current+state+of+the+science.">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175 (2016).</li><li>Gr&#252;n, D., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+analysis+of+single-cell+sequencing+experiments.">Design and analysis of single-cell sequencing experiments.</a> Cell. 163 (4), 799-810 (2015).</li><li>Ziegenhain, C., 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=Comparative+analysis+of+single-cell+RNA+sequencing+methods.">Comparative analysis of single-cell RNA sequencing methods.</a> Molecular cell. 65 (4), 631-643 (2017).</li><li>Hashimshony, 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=CEL-Seq2:+sensitive+highly-multiplexed+single-cell+RNA-Seq.">CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq.</a> Genome Biology. 17 (1), 77 (2016).</li><li>Islam, S., 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=Characterization+of+the+single-cell+transcriptional+landscape+by+highly+multiplex+RNA-seq.">Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq.</a> Genome Research. 21 (7), 1160-1167 (2011).</li><li>Macosko, E. Z., 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=Highly+parallel+genome-wide+expression+profiling+of+individual+cells+using+nanoliter+droplets.">Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets.</a> Cell. 161 (5), 1202-1214 (2015).</li><li>Klein, A. M., 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=Droplet+barcoding+for+single-cell+transcriptomics+applied+to+embryonic+stem+cells.">Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.</a> Cell. 161 (5), 1187-1201 (2015).</li><li>. Library Prep -Single Cell Gene Expression -Official 10x Genomics Support Available from: <a target="_blank" href="https://support.10xgenomics.com/single-cell-gene-expression/library-prep">https://support.10xgenomics.com/single-cell-gene-expression/library-prep</a> (2018)</li><li>McGinnis, C. S., 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=MULTI-seq:+sample+multiplexing+for+single-cell+RNA+sequencing+using+lipid-tagged+indices.">MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices.</a> Nature Methods. 1, (2019).</li><li>Stoeckius, M., 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=Cell+hashing+with+barcoded+antibodies+enables+multiplexing+and+doublet+detection+for+single+cell+genomics.">Cell hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics.</a> Genome Biology. 19 (1), 224 (2018).</li><li>Chan, M. M., 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=Molecular+recording+of+mammalian+embryogenesis.">Molecular recording of mammalian embryogenesis.</a> Nature. 570 (7759), 77-82 (2019).</li><li>. Agilent 4200 TapeStation System Available from: <a target="_blank" href="https://www.agilent.com/cs/library/datasheets/public/5991-6029EN.pdf">https://www.agilent.com/cs/library/datasheets/public/5991-6029EN.pdf</a> (2019)</li><li>. SPRIselect User Guide Available from: <a target="_blank" href="https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/SPRIselect%20User%20Guide.pdf">https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/SPRIselect%20User%20Guide.pdf</a> (2012)</li><li>. Qubit 4 Fluorometer User Guide Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf</a> (2018)</li><li>. Agilent High Sensitivity DNA Kit Guide Available from: <a target="_blank" href="https://www.agilent.com/cs/library/usermanuals/public/High%20Sensitivity_DNA_KG.pdf">https://www.agilent.com/cs/library/usermanuals/public/High%20Sensitivity_DNA_KG.pdf</a> (2016)</li><li>Butler, A., Hoffman, P., Smibert, P., Papalexi, E., Satija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+single-cell+transcriptomic+data+across+different+conditions,+technologies,+and+species.">Integrating single-cell transcriptomic data across different conditions, technologies, and species.</a> Nature Biotechnology. 36 (5), 411 (2018).</li><li>Weber, R. J., Liang, S. I., Selden, N. S., Desai, T. A., Gartner, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+fatty-acid+modified+oligonucleotides+to+live+cell+membranes+through+stepwise+assembly.">Efficient targeting of fatty-acid modified oligonucleotides to live cell membranes through stepwise assembly.</a> Biomacromolecules. 15 (12), 4621-4626 (2014).</li><li>. Single Cell Protocols Cell Preparation Guide Available from: <a target="_blank" href="https://support.10xgenomics.com/single-cell-gene-expression/sample-prep">https://support.10xgenomics.com/single-cell-gene-expression/sample-prep</a> (2017)</li></ol>

In this study, we have demonstrated a protocol to analyze single cell transcriptional profiles. We have also provided two optional methods to multiplex samples in the scRNA-Seq workflow. Both methods have proved to be feasible at various labs and provided solutions to run a cost-effective and batch effect-free single cell experiment18,26.
There are a few steps that should be followed carefully when going through the protocol. An ideal ...

The transcriptional profile of each single cell varies among cell populations during embryonic development. Although single molecular in situ hybridization can be used to visualize the expression of a small number of genes1, single cell mRNA sequencing (scRNA-Seq) provides an unbiased approach to illustrate genome-wide expression patterns of genes in single cells. After it was first published in 20092, scRNA-Seq has been applied to study multiple tissues at multiple developmental stages in the recent years3,4,5. Also, as the human cell atlas has launched its developmental-focused projects recently, more single cell data from human embryonic tissues are expected to be generated in the near future.
The heart as the first organ to develop plays a critical role in embryonic development. The heart consists of multiple cell types and the development of each cell type is tightly regulated temporally and spatially. Over the past few years, the origin and cell lineage of cardiac cells at early developmental stages have been characterized6, which provide a tremendous useful navigation tool for understanding congenital heart disease pathogenesis, as well as for developing more technologically advanced methods to stimulate cardiomyocyte regeneration7.
The scRNA-Seq has undergone a rapid expansion in recent years8,9,10. With the newly developed methods, design and analysis of single cell experiments has become more achievable11,12,13,14. The method presented here is a commercial procedure based on the droplet solutions (see Table of Materials)15,16. This method features capturing cells and sets of uniquely barcoded beads in an oil-water emulsion droplet under control of a microfluidic controller system. The rate of cell loading into the droplets is extremely low so that the majority of droplet emulsions contain only one cell17. The procedure&#39;s ingenious design comes from single cell separation into droplet emulsions occurring simultaneously with barcoding, which enables the parallel analysis of individual cells using RNA-Seq on a heterogeneous population.
The incorporation of multiplexing strategies is one of the important additions to the traditional single cell workflow13,14. This addition is very useful in discarding cell doublets, reducing experimental costs, and eliminating batch effects18,19. A lipid based barcoding strategy and an antibody based barcoding strategy (see Table of Materials) are the two mostly used multiplexing methods. Specific barcodes are used to label each sample in both methods, and the labeled samples are then mixed for single cell capturing, library preparation, and sequencing. Afterwards, the pooled sequencing data can be separated by analyzing the barcode sequences (Figure 1)19. However, significant differences exist between the two methods. The lipid based barcoding strategy is based on lipid-modified oligonucleotides, which has not been found to have any cell type preferences. While the antibody based barcoding strategy can only detect the cells expressing the antigen proteins19,20. In addition, it takes about 10 min to stain the lipids but 40 min to stain the antibodies (Figure 1). Furthermore, the lipid-modified oligonucleotides are cheaper than antibody-conjugated oligonucleotides but not commercially available at the time of writing this article. Finally, the lipid-based strategy can multiplex 96 samples in one experiment, but the antibody-based strategy currently can only multiplex 12 samples.
The recommended cell number to multiplex in a single experiment should be lower than 2.5 x 104, otherwise, it will lead to a high percentage of cell doublets and potential ambient mRNA contamination. Through the multiplexing strategies, the cost of single cell capturing, cDNA generation, and library preparation for multiple samples will be reduced to the cost of one sample but the sequencing cost will remain the same.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Tween-20</td>
			<td>Bio-Rad</td>
			<td>1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Chip Holder</td>
			<td>10x Genomics</td>
			<td>120252 330019</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Chromium Controller</td>
			<td>10x Genomics</td>
			<td>120223</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Magnetic Separator</td>
			<td>10x Genomics</td>
			<td>120250 230003</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Vortex Adapter</td>
			<td>10x Genomics</td>
			<td>330002, 120251</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Vortex Clip</td>
			<td>10x Genomics</td>
			<td>120253 230002</td>
			<td></td>
		</tr>
		<tr>
			<td>4200 TapeStation System</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td>University of Pittsburgh Health Sciences Sequencing Core</td>
		</tr>
		<tr>
			<td>Barcode Oligo</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>25 nmol</td>
		</tr>
		<tr>
			<td>Buffer EB</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1 mice</td>
			<td>Chales River</td>
			<td>Strain Code 022</td>
			<td>ordered pregnant mice</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Appendorf</td>
			<td>2231000214</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Chip B Single Cell Kit, 48 rxns</td>
			<td>10x Genomics</td>
			<td>1000073</td>
			<td>Store at ambient temperature</td>
		</tr>
		<tr>
			<td>Chromium i7 Multiplex Kit, 96 rxns</td>
			<td>10x Genomics</td>
			<td>120262</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; GEM Kit v3,4 rxns</td>
			<td>10x Genomics</td>
			<td>1000094</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; Library Kit v3</td>
			<td>10x Genomics</td>
			<td>1000095</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; v3 Gel Beads</td>
			<td>10x Genomics</td>
			<td>2000059</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Sigma/Millipore</td>
			<td>10103578001</td>
			<td>Store powder at 4 &#176;C, store at -20 &#176;C after it dissolves</td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Sigma/Millipore</td>
			<td>11088807001</td>
			<td>Store powder at 4 &#176;C, store at -20 &#176;C after it dissolves</td>
		</tr>
		<tr>
			<td>D1000 ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td>University of Pittsburgh Health Sciences Sequencing Core</td>
		</tr>
		<tr>
			<td>DNA LoBind Tube Microcentrifuge Tube, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>022431021</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tube Microcentrifuge Tube, 2.0 mL</td>
			<td>Eppendorf</td>
			<td>022431048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne SILANE</td>
			<td>10x Genomics</td>
			<td>2000048</td>
			<td>Store at 4 &#176;C, used in Beads Cleanup Mix (Table 1)</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Theromo Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, Pure (200 Proof, anhydrous)</td>
			<td>Sigma</td>
			<td>E7023-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15mL High Clarity PP Centrifuge Tube</td>
			<td>Corning Cellgro</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL High Clarity PP Centrifuge Tube</td>
			<td>Corning Cellgro</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, United States</td>
			<td>Fisher Scientific</td>
			<td>26140079</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Finnpipette F1 Multichannel Pipettes, 10-100&#956;l</td>
			<td>Theromo Scientific</td>
			<td>4661020N</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F1 Multichannel Pipettes, 1-10&#956;l</td>
			<td>Theromo Scientific</td>
			<td>4661000N</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmi Cell Strainer</td>
			<td>Sigma</td>
			<td>BAH136800040</td>
			<td>Porosity 40 &#956;m, for 1000 uL Pipette Tips, pack of 50 each</td>
		</tr>
		<tr>
			<td>Glycerin (Glycerol), 50% (v/v)</td>
			<td>Ricca Chemical Company</td>
			<td>3290-32</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX (Fc Receptor Blocking Solution)</td>
			<td>BioLegend</td>
			<td>422301</td>
			<td>Add 5 &#181;l of Human TruStain FcX per million cells in 100 &#181;l staining volume</td>
		</tr>
		<tr>
			<td>Isopropanol (IPA)</td>
			<td>Fisher Scientific</td>
			<td>A464-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa HiFi HotStart ReadyMix (2X)</td>
			<td>Fisher Scientific</td>
			<td>NC0295239</td>
			<td>Store at -20 &#176;C, used in Lipid-tagged barcode library mix (Table 1)</td>
		</tr>
		<tr>
			<td>Lipid Barcode Primer (Multi-seq Primer)</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>100 nmol</td>
		</tr>
		<tr>
			<td>Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090-015</td>
			<td></td>
		</tr>
		<tr>
			<td>MasterCycler Pro</td>
			<td>Eppendorf</td>
			<td>950W</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (Ambion)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tubes 0.2 ml 8-tube strips</td>
			<td>Eppendorf</td>
			<td>951010022</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1X without calcium &#38; magnesium</td>
			<td>Corning Cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) with 10% Bovine Albumin (alternative to Thermo Fisher product)</td>
			<td>Sigma-Aldrich</td>
			<td>SRE0036</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 4-pack (0.1&#8211;2.5&#956;L, 0.5-10&#956;L, 10&#8211;100&#956;L, 100&#8211;1,000&#956;L variable-volume pipettes</td>
			<td>Fisher Scientific</td>
			<td>05-403-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection reagent (SPRIselect Reagent Kit)</td>
			<td>Beckman Coulter</td>
			<td>B23318 (60ml)</td>
			<td></td>
		</tr>
		<tr>
			<td>Template Switch Oligo</td>
			<td>10x Genomics</td>
			<td>3000228</td>
			<td>Store at -20 &#176;C, used in Master Mix (Table 1)</td>
		</tr>
		<tr>
			<td>The antibody based barcoding strategy is also known as Cell Hashing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The cell browser is Loup Cell Browser</td>
			<td>10x Genomics</td>
			<td></td>
			<td><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser" target="_blank">https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser</a></td>
		</tr>
		<tr>
			<td>The commercial available analysis pipline in step 8.1 is Cell Ranger</td>
			<td>10x Genomics</td>
			<td></td>
			<td><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger" target="_blank">https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger</a></td>
		</tr>
		<tr>
			<td>The lipid based barcoding strategy is also known as MULTI-seq</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The well maintained R platform is Seurat V3</td>
			<td>satijalab</td>
			<td></td>
			<td><a href="https://satijalab.org/seurat/" target="_blank">https://satijalab.org/seurat/</a></td>
		</tr>
		<tr>
			<td>TipOne RPT 0.1-10/20 ul XL ultra low retention filter pipet tip</td>
			<td>USA Scientific</td>
			<td>1180-3710</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne RPT 1000 ul XL ultra low retention filter pipet tip</td>
			<td>USA Scientific</td>
			<td>1182-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne RPT 200 ul ultra low retention filter pipet tip</td>
			<td>USA Scientific</td>
			<td>1180-8710</td>
			<td></td>
		</tr>
		<tr>
			<td>TotalSeq-A0301 anti-mouse Hashtag 1 Antibody</td>
			<td>BioLegend</td>
			<td>155801</td>
			<td>0.1 - 1.0 &#181;g of antibody in 100 &#181;l of staining buffer for every 1 million cells</td>
		</tr>
		<tr>
			<td>TotalSeq-A0302 anti-mouse Hashtag 2 Antibody</td>
			<td>BioLegend</td>
			<td>155803</td>
			<td>0.1 - 1.0 &#181;g of antibody in 100 &#181;l of staining buffer for every 1 million cells</td>
		</tr>
		<tr>
			<td>TotalSeq-A0302 anti-mouse Hashtag 3 Antibody</td>
			<td>BioLegend</td>
			<td>155805</td>
			<td>0.1 - 1.0 &#181;g of antibody in 100 &#181;l of staining buffer for every 1 million cells</td>
		</tr>
		<tr>
			<td>TrueSeq RPI primer</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>100 nmol, used in Lipid-tagged barcode library mix (Table 1)</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Fisher Scientific</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal I5</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>100 nmol</td>
		</tr>
	</tbody>
</table>

multiplexed single cell mrna sequencing analysis of mouse embryonic cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

In this study, we used mouse embryonic heart as an example to exhibit how multiplexed single cell mRNA sequencing was performed to process the different samples from separate parts of an organ simultaneously. E18.5 CD1 mouse hearts were isolated and dissected into left atrium (LA), right atrium (RA), left ventricle (LV) and right ventricle (RV). The atrial and ventricular cells were then barcoded independently using a lipid-based barcoding procedure and mixed together before GEMs generati...

Watch this Scientific Journal Video about Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells at JoVE.com

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Single cell mRNA sequencing is widely used to understand the biological processes at a single cell level. The advent of multiplexing strategies has further expanded its amplifications by allowing the profiling of multiple samples in a single experiment, dramatically reducing the cost and avoiding the batch effects. Demonstrating the procedure will be Wei Feng, a postdoc, and Andrew Przysinda, a technician from my lab.After counting, split the cells isolated from embryonic day 18.5 heart chambers into five times 10 to the fifth aliquots and wash the cells two times with fresh PBS per wash. Resuspend the pellets in 180 microliters of PBS per sample and add 20 microliters of anchor barcode stock solution to each tube with gentle mixing. After a five-minute incubation on ice, add 20 microliters of co-anchor barcode stock solution to each tube with gentle mixing for an additional five-minute incubation on ice before washing the cells three times with one milliliter of cold PBS supplemented with 1%bovine serum albumin per wash, then filter the samples into new tubes through one 40-micrometer cell strainer per tube.After counting, assemble chip B into a chip holder, then add 75 microliters of a 50%glycerol solution to the unused wells in row one, 40 microliters to the unused wells in row two, and 280 microliters to the unused wells in row three. Note that a training chip is used here to demonstrate the procedure. Add the appropriate volume of cell suspension and nuclease-free water to the master mix with gentle pipetting.Add 75 microliters of the resulting cell solution to the bottom center of the sample well in row one without bubbles. Vortex the gel beads for 30 seconds before slowly adding 40 microliters of beads to the bottom center of the gel bead well in row two without bubbles. Add 280 microliters of partitioning oil down the sidewall of the partitioning oil well in row three.Attach the gasket to the chip without pressing down on the gasket and keeping the gasket horizontal to avoid wetting the gasket. Load the assembled chip with the gasket in the chromium controller and run the chromium single cell B program. Note that the screen shows chromium training for the training chip but for the real experiments the screen will display chromium single cell B.When the program has completed, immediately remove the chip and discard the gasket.Fold the lid back to expose the wells at a 45-degree angle and check the liquid levels to make sure no clogs are present. Slowly aspirate 100 microliters of gel beads in emulsion from the lowest points of the recovery well and check the uniformity of the beads. Dispense the emulsified beads down the wall of a new PCR tube on ice and place the tube in a thermal cycler for reverse transcription.To prepare the cDNA for amplification, add 125 microliters of recovery agent to the sample at room temperature. No opaque liquid should be observed and avoid pipetting or vortexing. Wait 60 seconds before slowly removing the recovery agent from the bottom of the tube and add 200 microliters of vortex bead cleanup mixture to the sample.Pipette the mixture 10 times before incubating for 10 minutes at room temperature. Next, place the samples onto a magnet in the high position. When the solution clears, remove the supernatant and add 200 microliters of 80%ethanol to the pellet.After 30 seconds, remove the ethanol and repeat the wash two more times. After the last wash, briefly centrifuge the sample and place the tube on the magnet in the low position to allow the sample to air dry for less than two minutes. When the sample has dried, remove the tube from the magnet and add 35.5 microliters of freshly prepared elution solution.After a two-minute incubation at room temperature, place the sample on the magnet in the high position until the solution clears before transferring 35 microliters of the sample to a new tube strip. For cDNA amplification using the lipid-based barcoding strategy, add amplification reaction mixture to the 35-microliter cDNA samples with thorough mixing before briefly centrifuging. At the end of the spin, incubate the sample in the thermal cycler following the appropriate cDNA amplification procedure.Add 120 microliters of select reagent and 100 microliters of ultrapure water to 100 microliters of sample and pipette the resulting 0.6x select reagent 15 times. After a five-minute incubation at room temperature, place the sample on the magnet until the solution becomes clear. Transfer the supernatant to a 1.5-milliliter low bind tube for multiplexing barcoded cDNA library construction.Remember to save both the supernatants and the base as the supernatant's contents assemble barcoded cDNA and the base contents endogenous cDNA. Clean the endogenous cDNA with 80%ethanol and elute the DNA with 40 microliters of elution buffer, then run a quality control check of the endogenous cDNA before preparing an endogenous transcript library. The cDNA concentration should be quantified and qualified before library construction.The constructed libraries, including the endogenous cDNA and barcode libraries, should also be quantified and qualified before sequencing. After sequencing, the barcode expression can be analyzed in each single cell. For example, in this analysis eight groups of single cells that uniquely expressed one type of barcode representing cells from eight different samples were observed.In addition, some cells did not express any barcode and therefore were defined as negative cells, while other cells expressed two different barcodes representing doublets. Using the singlet cells, the cellular heterogeneity and molecular regulations could be assessed by cell type annotation, novel and rare cell type identification, anatomical zone comparative analysis, and gene ontology pathway analysis, such as cell cycle phase separations. It is helpful to image the gel beads in emulsion if you can, as an image will tell you whether or not the procedure has been successful up to this point.The sample multiplexing adds great flexibility in designing your experiments. After watching this demonstration, I hope you will be more confident about starting your own experiments.

multiplexed-single-cell-mrna-sequencing-analysis-mouse-embryonic

Research

JoVE Journal

Developmental Biology

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

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 ...

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (&#8804;200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens ...

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 ...

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.