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.

<ol>
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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. 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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=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>

Nothing to disclose.

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>
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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 ...

multiplexed-analysis-retinal-gene-expression-chromatin-accessibility

Research

JoVE Journal

Neuroscience

3.4K Views.  Johns Hopkins University School of Medicine. 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.

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

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 a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

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 environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

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 ...

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 nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

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 the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

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

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 this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

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

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

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

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 basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

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

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

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 ...

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 microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

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 wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

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

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...