Name: multiplexed analysis of retinal gene expression and chromatin accessibility using scrna-seq and scatac-seq
Uploaded: 2021-03-12T13:30:00
Description: Watch this Scientific Journal Video about Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq at JoVE.com

We thank Linda Orzolek from the Johns Hopkins Transcriptomics and Deep Sequencing Core for help in sequencing the produced libraries and Lizhi Jiang for performing the ex vivo retinal explants.

The use of animals for these studies was conducted using protocols approved by the Johns Hopkins Animal Care and Use Committee, in compliance with ARRIVE guidelines, and were performed in accordance with relevant guidelines and regulations.
1. MULTI-seq
<ol>
	<li>Media preparation
	<ol>
		<li>Prepare and equilibrate ovomucoid inhibitor, 10 mg of ovomucoid inhibitor and 10 mg of albumin per mL of Earle&#39;s Balanced Salt Solution (EBSS), for 30 min prior to use<sup class="xr...

<ol>
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H., Bang, D. <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+RNA+sequencing+technologies+and+bioinformatics+pipelines.">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Experimental &amp; Molecular Medicine. 50, 96 (2018).</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, 411-420 (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. 16, 619-626 (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, 224 (2018).</li><li>Chen, X., Miragaia, R. J., Natarajan, K. N., Teichmann, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+robust+method+for+single+cell+chromatin+accessibility+profiling.">A rapid and robust method for single cell chromatin accessibility profiling.</a> Nature Communications. 9, 5345 (2018).</li><li>Hoang, 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=Gene+regulatory+networks+controlling+vertebrate+retinal+regeneration.">Gene regulatory networks controlling vertebrate retinal regeneration.</a> Science. , 8598 (2020).</li><li>Clark, B. 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=Single-Cell+RNA-Seq+Analysis+of+Retinal+Development+Identifies+NFI+Factors+as+Regulating+Mitotic+Exit+and+Late-Born+Cell+Specification.">Single-Cell RNA-Seq Analysis of Retinal Development Identifies NFI Factors as Regulating Mitotic Exit and Late-Born Cell Specification.</a> Neuron. 102, 1111-1126 (2019).</li><li>Zheng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+circulating+immune+cell+landscape+in+aging+and+COVID-19.">A human circulating immune cell landscape in aging and COVID-19.</a> Protein Cell. 11, 740-770 (2020).</li><li>Satpathy, A. 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=Massively+parallel+single-cell+chromatin+landscapes+of+human+immune+cell+development+and+intratumoral+T+cell+exhaustion.">Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion.</a> Nature Biotechnology. 37, 925-936 (2019).</li><li>Worthington Biochemical Corporation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Worthington Biochemical Corporation. Papain Dissociation System. , (2020).</li><li>10x Genomics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromium Single Cell 3' Reagent Kits v3 User Guide. , (2020).</li><li>Agilent. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DNF-468 HS Genomic DNA 50 kb Kit Quick Guide for Fragment Analyzer Systems. , (2015).</li><li>ThermoFisher Scientific. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qubit+dsDNA+HS+Assay+Kits.">Qubit dsDNA HS Assay Kits.</a> ThermoFisher Scientific. , (2015).</li><li>10x Genomics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromium Single Cell ATAC Reagent Kits User Guide (v1.1 Chemistry). , (2020).</li><li>Weir, K., Kim, D. W., Blackshaw, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+retinal+neurogenesis+by+somatostatin+signaling.">Regulation of retinal neurogenesis by somatostatin signaling.</a> bioRxiv. , (2020).</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=Simultaneous+epitope+and+transcriptome+measurement+in+single+cells.">Simultaneous epitope and transcriptome measurement in single cells.</a> Nature Methods. 14, 865-868 (2017).</li><li>Gaublomme, J. 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=Nuclei+multiplexing+with+barcoded+antibodies+for+single-nucleus+genomics.">Nuclei multiplexing with barcoded antibodies for single-nucleus genomics.</a> Nature Communications. 10, 2907 (2019).</li><li>Ma, 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=Chromatin+Potential+Identified+by+Shared+Single-Cell+Profiling+of+RNA+and+Chromatin.">Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin.</a> Cell. , (2020).</li><li>Buenrostro, J. D., 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=Integrated+Single-Cell+Analysis+Maps+the+Continuous+Regulatory+Landscape+of+Human+Hematopoietic+Differentiation.">Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation.</a> Cell. 173, 1535-1548 (2018).</li><li>Pliner, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cicero+Predicts+cis-Regulatory+DNA+Interactions+from+Single-Cell+Chromatin+Accessibility+Data.">Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data.</a> Molecular Cell. 71, 858-871 (2018).</li><li>Granja, J. 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=ArchR:+An+integrative+and+scalable+software+package+for+single-cell+chromatin+accessibility+analysis.">ArchR: An integrative and scalable software package for single-cell chromatin accessibility analysis.</a> BioRxiv. , (2020).</li><li>Stuart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Integration+of+Single-Cell+Data.">Comprehensive Integration of Single-Cell Data.</a> Cell. 177, 1888-1902 (2019).</li><li>METABRIC Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+and+transcriptomic+architecture+of+2,000+breast+tumours+reveals+novel+subgroups.">The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.</a> Nature. 486, 346-352 (2012).</li><li>Izadi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Connectivity+in+Colorectal+Cancer+Gene+Expression+Network.">Differential Connectivity in Colorectal Cancer Gene Expression Network.</a> Iranian Biomedical Journal. 23, 34-46 (2019).</li></ol>

The power of MULTI-seq stems from seamless integration of data from multiple experimental conditions or models and the enormous benefit in terms of cost and limiting batch effects. Utilizing MULTI-seq offers a laboratory unprecedented phenotyping depth. Non-genetic multiplexing methods such as cell hashing or nuclei hashing opened the door to multiplexed samples through the use of barcoded antibodies7,19,20. However, this relies...

Understanding how genes can influence cell fate plays a key role in interrogating processes such as disease and embryonic progression. The complex relationships between transcription factors and their target genes can be grouped in gene regulatory networks. Mounting evidence places these gene regulatory networks at the center of both disease and development across evolutionary lineages1. While previous techniques such as qRT-PCR focused on a single gene or set of genes, the application of high-throughput sequencing technology allows for the profiling of complete cellular transcriptomes.
RNA-seq offers a glimpse into large scale transcriptomics2,3. Single-cell RNA sequencing (scRNA-seq) gives investigators the ability to not only profile transcriptomes but link specific cell types with gene expression profiles4. This is achieved bioinformatically by feeding individual cell profiles into sorting algorithms using known gene markers5. Multiplexing using lipid-tagged indices sequencing (MULTI-seq) offers unprecedented diversity in the number of scRNA-Seq profiles that can be collected6. This lipid based technique differs from other sample indexing techniques such as cell-hashing that rely on the presence of surface antigens and high affinity antibodies instead of plasma membrane integration7. Not only is it now possible to profile gene expression profiles into cell types but different experiments can be combined into a single sequencing library, dramatically lowering the cost of an individual scRNA-seq experiment6. The cost of scRNA-seq may seem prohibitive for use in phenotyping experiments where many different genotypes, conditions or patient samples are analyzed, but multiplexing allows the combination of up to 96 samples in a single library6.
Profiling gene expression via scRNA-seq has not been the only high-throughput sequencing-based technique to revolutionize the current understanding of how molecular mechanisms dictate cell fate. While understanding which gene transcripts are present in a cell enables the identification of cell type, equally important is understanding how genomic organization regulates development and disease progression. Early studies relied on detecting DNase-mediated cleavage of sequences not bound to histones, followed by sequencing of the resulting DNA fragments to identify regions of open chromatin. In contrast, single cell assay for transposon accessible chromatin sequencing (scATAC-seq) allows researchers to probe DNA with a domesticated transposon to readily profile open chromatin at the single nucleotide level8. This has gone through a similar scaling to scRNA-seq and now investigators can identify individual cell types and profile phenotypes across thousands of individual genomes8.
The pairing of scRNA-seq and scATAC-seq has allowed researchers the ability to profile thousands of cells to determine cell populations, genomic organization, and gene regulatory networks in disease models and developmental processes9,10,11,12. Here the authors outline how to first utilize MULTI-seq to condense phenotyping of a myriad of animal models and employ paired scRNA-seq and scATAC-seq to gain a better understanding of the chromatin landscape and regulatory networks in these animal models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#181;L, 200 &#181;L, 1000 &#181;L pipette filter tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>Bio-Rad</td>
			<td>1662404</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;M Barcode Solution</td>
			<td>Request from Gartner lab</td>
			<td>https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP 
			rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&#38;edit_requested 
			=true</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Millipore Sigma</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Methanol</td>
			<td>Millipore Sigma</td>
			<td>322415-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Chip Holder</td>
			<td>10x Genomics</td>
			<td>1000195</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Chromium controller &#38; Accessory Kit</td>
			<td>10x Genomics</td>
			<td>PN-120263</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Centrifuge Tube</td>
			<td>Quality Biological</td>
			<td>P886-229411</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m FlowMi Cell Strainer</td>
			<td>Bel-Art</td>
			<td>H13680-0040</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;M Anchor Solution</td>
			<td>Sigma or request from Gartner lab</td>
			<td>https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP 
			rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&#38;edit_requested 
			=true</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;M Co-Anchor Solution</td>
			<td>Sigma or request from Gartner lab</td>
			<td>https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP 
			rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&#38;edit_requested 
			=true</td>
			<td></td>
		</tr>
		<tr>
			<td>5200 Fragment Analyzer system</td>
			<td>Agilent</td>
			<td>M5310AA</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um FlowMi cell strainer</td>
			<td>Bel-Art</td>
			<td>H13680-0070</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-12R Centrifuge</td>
			<td>VWR</td>
			<td>BK392302</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Chip G</td>
			<td>10x Genomics</td>
			<td>PN-1000120</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next GEM Chip H</td>
			<td>10x Genomics</td>
			<td>PN-1000161</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Next Gem Single Cell ATAC Reagent Kit v1.1</td>
			<td>10x Genomics</td>
			<td>PN-1000175</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; GEM, Library &#38; Gel Bead Kit v3.1</td>
			<td>10x Genomics</td>
			<td>PN-1000121</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Fisher Scientific</td>
			<td>BN2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EVA Foam Ice Pan</td>
			<td>Tequipment</td>
			<td>04393-54</td>
			<td></td>
		</tr>
		<tr>
			<td>FA 12-Capillary Array Short, 33 cm</td>
			<td>Agilent</td>
			<td>A2300-1250-3355</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Isotemp Water Bath</td>
			<td>Fisher Scientific</td>
			<td>15-460-20Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Forma CO2 Water Jacketed Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol 50% Aqueous solution</td>
			<td>Ricca Chemical Company</td>
			<td>3290-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Hausser Scientific Bright-Line Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina NextSeq or NovaSeq</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa Hifi Hotstart ReadyMix</td>
			<td>HiFi</td>
			<td>7958927001</td>
			<td></td>
		</tr>
		<tr>
			<td>Low TE Buffer</td>
			<td>Quality Biological</td>
			<td>351-324-721</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution 1 M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Separator Rack for 1.5 mL tubes</td>
			<td>Millipore Sigma</td>
			<td>20-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Separator Rack for 200 &#181;L tubes</td>
			<td>10x Genomics</td>
			<td>NC1469069</td>
			<td></td>
		</tr>
		<tr>
			<td>MULTI-seq Primer</td>
			<td>Sigma or IDT</td>
			<td>See sequence list</td>
			<td></td>
		</tr>
		<tr>
			<td>MyFuge Mini Centrifuge</td>
			<td>Benchmark Scientific</td>
			<td>C1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonidet P40 Substitute</td>
			<td>Sigma-Aldrich</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Fisher Scientific</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>P2, P10, P20, P200, P1000 micropipettes</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (1X)</td>
			<td>Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiagen Buffer EB</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>Refridgerated Centrifuge 5424 R</td>
			<td>Eppendorf</td>
			<td>2231000655</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free Disposable Pellet Pestles</td>
			<td>Fisher Scientific</td>
			<td>12-141-368</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Plus RNase Inhibitor</td>
			<td>Promega</td>
			<td>N2615</td>
			<td></td>
		</tr>
		<tr>
			<td>RPI primer</td>
			<td>Sigma or IDT</td>
			<td>See sequence list</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Index Kit N, Set A</td>
			<td>10x Genomics</td>
			<td>PN-1000212</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Index Kit T Set A</td>
			<td>10x Genomics</td>
			<td>PN-1000213</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride Solution 5 M</td>
			<td>Sigma-Aldrich</td>
			<td>59222C</td>
			<td></td>
		</tr>
		<tr>
			<td>SPRIselect Reagent Kit</td>
			<td>Beckman Coulter</td>
			<td>B23318</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Disposable Transfer Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>TempAssure PCR 8-tube strip</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Hydrochloride Solution, pH 7.4</td>
			<td>Sigma-Aldrich</td>
			<td>T2194</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4% (w/v)</td>
			<td>Corning</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal I5 primer</td>
			<td>Sigma or IDT</td>
			<td>See sequence list</td>
			<td></td>
		</tr>
		<tr>
			<td>Veriti Thermal Cycler</td>
			<td>Applied Biosystems</td>
			<td>4375786</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>VWR</td>
			<td>10153-838</td>
			<td></td>
		</tr>
	</tbody>
</table>

multiplexed analysis of retinal gene expression and chromatin accessibility using scrna-seq and scatac-seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

This workflow lays out a strategy for investigation of developmental phenotypes and regulatory processes using single cell sequencing. MULTI-seq sample multiplexing enables an initial low-cost phenotyping assay while paired collection and fixation of samples for scRNA-seq and scATAC-seq allows for more in-depth investigation (Figure 1).
MULTI-seq barcoding enables the combined sequencing of multiple samples and their subsequent computational deconvolution. The sam...

Watch this Scientific Journal Video about Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq at JoVE.com

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

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.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

This protocol will enable the use of single-cell RNA-seq for initial phenotyping. And, when paired with single-cell ATAC-seq, the reconstruction of gene regulatory networks. The use of MULTI-seq keeps costs low for initial investigation, and fixing paired samples for single-cell RNA-seq and single-cell ATAC-seq allows for time course analysis of gene regulatory networks.Investigators can utilize this protocol to understand the genetic networks controlling development and disease progression. To begin, add 20 microliters of anchor barcode solution to each sample and pipette it to mix. Incubate it on ice for five minutes.Then add 20 microliters of co-anchor solution to each sample, and incubate on ice for another five minutes. Add one milliliter of ice cold 1%BSA and PBS to each sample and centrifuge the cells at 300 x g for five minutes at four degree Celsius. Remove the supernatant without disturbing the cell pellet.Then add 400 microliters of ice cold 1%BSA and PBS. Centrifuge at 300 x g for five minutes at four degree Celsius. Determine the concentration of cells for each sample using a hemocytometer, and calculate the total number of cells and volume required for Gel Bead-in-Emulsion or GEM.Combine the calculated volumes from each sample in a new sterile 1.5 milliliter tube, and determine the final cell concentration of the combined samples. After GEM generation and barcoding, perform gene expression cDNA cleanup using size selection, making sure not to discard the supernatant from the first round of selection, which contains the sample barcode. Transfer the supernatant to a new sterile 1.5 milliliter microcentrifuge tube and store it on ice.Vortex the paramagnetic bead-based size selection reagent, and add 260 microliters of the reagent and 180 microliters of 100%Isopropanol to the supernatant. Pipette the mix 10 times and incubate at room temperature for five minutes. Place the tube on a magnetic rack and wait for the solution to clear.Once the solution is clear, remove and discard the supernatant. Add 500 microliters of 80%ethanol to the beads for 30 seconds, then discard the supernatant. Repeat this for a total of two washes.Briefly centrifuge the beads and return them to the magnetic rack. Set the timer to two minutes. Then remove the residual ethanol with a P10 pipette, and air dry the beads on the magnetic rack for the remaining two minutes.Remove the beads from the magnetic rack and resuspend them in 100 microliters of elution buffer. Pipette thoroughly to resuspend, and incubate at room temperature for two minutes, then return the beads to the magnetic rack and wait for the solution to clear. Transfer the supernatant to a fresh 1.5 milliliter microcentrifuge tube.Repeat the cleanup as demonstrated, using half the volume of elution buffer. For the samples destined for scATAC sequence, remove any liquid from the microcentrifuge tube using a P200 pipette, and flash-freeze the samples in ethanol dry ice. To dissociate the cells in the samples destined for scRNA sequencing, perform the dissociation steps using papain, as described in the text manuscript.Centrifuge the cells at 300 x g for three minutes at room temperature, and remove the supernatant without disrupting the cell pellet. Add one milliliter of chilled PBS, and mix 10 times or until the cells are completely suspended. Again, centrifuge at 300 x g for five minutes at four degree Celsius, and repeat the wash with PBS twice.Start vortexing the tube at the lowest speed setting and add 900 microliters of chilled methanol, drop by drop, while continuing to gently vortex the tube to prevent the cells from clumping. Incubate the cells on ice for 15 minutes. Determine the concentration of the fixed cells using a hemocytometer, and assess the efficacy of the fixation step using trypan blue.Store the frozen tissue and fixed cells at minus 80 degrees Celsius. MULTI sequence barcoding enables the combined sequencing of multiple samples and their subsequent computational deconvolution. The sample of origin can be determined for each cell based on their barcode expression.These combined samples can be analyzed as a single dataset for the purposes of cell clustering and cell type identification. Because each cell is barcoded before GEM generation, cell doublets will have a high probability of showing expression for multiple MULTI sequence barcodes, and a majority of doublets can therefore be identified and removed prior to clustering and cell type identification. The single-cell ATAC sequencing can be used to generate a dataset with cell types to match those found by single-cell RNA sequencing.The pairing of single-cell RNA sequencing gene expression and single-cell ATAC sequencing DNA accessibility information enables the reconstruction of gene regulatory networks. When attempting the protocol, keep in mind that the two-minute timing of the ethnol evaporation is crucial for sequence retrieval in the subsequent elution steps. Reconstructing gene regulatory networks using paired single-cell RNA-seq and single-cell ATAC-seq produces targets for gene overexpression and knockout studies.MULTI-seq can be employed at this stage to identify cell type specific effects.

Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Mosaic Analysis of Gene Function in Postnatal Mouse Brain Development by Using Virus-based Cre Recombination

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal 
development when ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...