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 ...

Research

JoVE Journal

Developmental Biology

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

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 ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

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, ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

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 ...

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 ...

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, ...

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 ...

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 ...