Name: Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells (Video) | JoVE Journal
Uploaded: 2020-01-07T14:00:00.000+00:00
Duration: 8 min 30 s
Description: Learn multiplexed single cell mRNA sequencing to profile gene expression in mouse embryonic tissues using droplet-based scRNA-Seq. This method reduces costs and minimizes batch effects for developmental studies.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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).

<ol><li>Raj, A., Van Den Bogaard, P., Rifkin, S. A., Van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nature Methods. 5 (10), 877(2008).</li>
<li>Tang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mRNA-Seq+whole-transcriptome+analysis+of+a+single+cell%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">mRNA-Seq whole-transcriptome analysis of a single cell.</a> Nature Methods. 6 (5), 377(2009).</li>
<li>Li, G., Plonowska, K., Kuppusamy, R., Sturzu, A., Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+cardiovascular+lineage+descendants+at+single-cell+resolution%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Identification of cardiovascular lineage descendants at single-cell resolution.</a> Development. 142 (5), 846-857 (2015).</li>
<li>DeLaughter, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+resolution+of+temporal+gene+expression+during+heart+development%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">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/pubmed?term=((Single+cell+expression+analysis+reveals+anatomical+and+cell+cycle-dependent+transcriptional+shifts+during+heart+development%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+deployment+of+cell+lineages+that+form+the+mammalian+heart%5BTitle%5D)+AND+%22Nature+Reviews+Cardiology%22%5BJournal%5D)">The deployment of cell lineages that form the mammalian heart.</a> Nature Reviews Cardiology. 1, (2018).</li>
<li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+transcriptomics+reconstructs+fate+conversion+from+fibroblast+to+cardiomyocyte%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte.</a> Nature. 551 (7678), 100(2017).</li>
<li>Lafzi, A., Moutinho, C., Picelli, S., Heyn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tutorial%3A+guidelines+for+the+experimental+design+of+single-cell+RNA+sequencing+studies%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies.</a> Nature Protocols. 1, (2018).</li>
<li>The Tabula Muris Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.</a> Nature. 562 (7727), 367(2018).</li>
<li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+genome+sequencing%3A+current+state+of+the+science%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175(2016).</li>
<li>Grün, D., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+analysis+of+single-cell+sequencing+experiments%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Comparative+analysis+of+single-cell+RNA+sequencing+methods%5BTitle%5D)+AND+%22Molecular+cell%22%5BJournal%5D)">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/pubmed?term=((CEL-Seq2%3A+sensitive+highly-multiplexed+single-cell+RNA-Seq%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+the+single-cell+transcriptional+landscape+by+highly+multiplex+RNA-seq%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">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/pubmed?term=((Highly+parallel+genome-wide+expression+profiling+of+individual+cells+using+nanoliter+droplets%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Droplet+barcoding+for+single-cell+transcriptomics+applied+to+embryonic+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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. , 10x Genomics. https://support.10xgenomics.com/single-cell-gene-expression/library-prep (2018).</li>
<li>McGinnis, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MULTI-seq%3A+sample+multiplexing+for+single-cell+RNA+sequencing+using+lipid-tagged+indices%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">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/pubmed?term=((Cell+hashing+with+barcoded+antibodies+enables+multiplexing+and+doublet+detection+for+single+cell+genomics%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">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/pubmed?term=((Molecular+recording+of+mammalian+embryogenesis%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Molecular recording of mammalian embryogenesis.</a> Nature. 570 (7759), 77-82 (2019).</li>
<li>Agilent 4200 TapeStation System. , Agilent. https://www.agilent.com/cs/library/datasheets/public/5991-6029EN.pdf (2019).</li>
<li>SPRIselect User Guide. , Beckman Coulter. https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/SPRIselect%20User%20Guide.pdf (2012).</li>
<li>Qubit 4 Fluorometer User Guide. , Thermo Fisher Scientific. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf (2018).</li>
<li>Agilent High Sensitivity DNA Kit Guide. , Agilent. https://www.agilent.com/cs/library/usermanuals/public/High%20Sensitivity_DNA_KG.pdf (2016).</li>
<li>Butler, A., Hoffman, P., Smibert, P., Papalexi, E., Satija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrating+single-cell+transcriptomic+data+across+different+conditions%2C+technologies%2C+and+species%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Efficient+targeting+of+fatty-acid+modified+oligonucleotides+to+live+cell+membranes+through+stepwise+assembly%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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. , 10x Genomics. https://support.10xgenomics.com/single-cell-gene-expression/sample-prep (2017).</li>
</ol>

The authors have no conflicts of interest to disclose.

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><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 &amp;deg;C</td></tr><tr><td>Chromium Single Cell 3&#039; GEM Kit v3,4 rxns</td><td>10x Genomics</td><td>1000094</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Chromium Single Cell 3&#039; Library Kit v3</td><td>10x Genomics</td><td>1000095</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Chromium Single Cell 3&#039; v3 Gel Beads</td><td>10x Genomics</td><td>2000059</td><td>Store at -80 &amp;deg;C</td></tr><tr><td>Collagenase A</td><td>Sigma/Millipore</td><td>10103578001</td><td>Store powder at 4 &amp;deg;C, store at -20 &amp;deg;C after it dissolves</td></tr><tr><td>Collagenase B</td><td>Sigma/Millipore</td><td>11088807001</td><td>Store powder at 4 &amp;deg;C, store at -20 &amp;deg;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 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Finnpipette F1 Multichannel Pipettes, 10-100&amp;mu;l</td><td>Theromo Scientific</td><td>4661020N</td><td></td></tr><tr><td>Finnpipette F1 Multichannel Pipettes, 1-10&amp;mu;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 &amp;mu;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 &amp;micro;l of Human TruStain FcX per million cells in 100 &amp;micro;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 &amp;deg;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 &amp;amp; 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&amp;ndash;2.5&amp;mu;L, 0.5-10&amp;mu;L, 10&amp;ndash;100&amp;mu;L, 100&amp;ndash;1,000&amp;mu;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 &amp;deg;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>&lt;a href=&quot;https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser&quot; target=&quot;_blank&quot;&gt;https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser&lt;/a&gt;</td></tr><tr><td>The commercial available analysis pipline in step 8.1 is Cell Ranger</td><td>10x Genomics</td><td></td><td>&lt;a href=&quot;https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger&quot; target=&quot;_blank&quot;&gt;https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger&lt;/a&gt;</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>&lt;a href=&quot;https://satijalab.org/seurat/&quot; target=&quot;_blank&quot;&gt;https://satijalab.org/seurat/&lt;/a&gt;</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 &amp;micro;g of antibody in 100 &amp;micro;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 &amp;micro;g of antibody in 100 &amp;micro;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 &amp;micro;g of antibody in 100 &amp;micro;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

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...

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 ...

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

Research

JoVE Journal

Developmental Biology

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Single cell expression profiling allows the detailed gene expression analysis of individual cells.  We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

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.

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

This protocol combines flow cytometry and single-cell RNA sequencing to isolate and characterize microglial cell states in the cerebellum of early postnatal mouse brains. It uses enzymatic dissociation, Percoll centrifugation, and immunostaining to reveal microglia heterogeneity and improve understanding of their roles in cerebellar development and disease.

Flow Cytometry and Single-Cell Analysis for Characterizing Microglia Activation in Early Postnatal Mouse Brain Development

This protocol provides a streamlined computational pipeline for quantifying nascent enhancer transcripts. By integrating chromatin accessibility, chromatin feature, and transcriptional data, it enables accurate detection and strand-specific analysis of enhancer activity in complex intragenic regions, while remaining accessible to researchers without extensive bioinformatics training.

A Computational Pipeline for Intergenic/Intragenic Enhancer RNA Quantification in Mouse Embryonic Stem Cells

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

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).

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

Learn multiplexed single cell mRNA sequencing to profile gene expression in mouse embryonic tissues using droplet-based scRNA-Seq. This method reduces costs and minimizes batch effects for developmental studies.

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Explore More Videos