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

我们感谢约翰霍普金斯大学转录组学和深度测序核心的 Linda Orzolek 帮助对产生的文库进行测序，并感谢蒋立志进行 离体 视网膜外植体。

这些研究中的动物使用是使用约翰霍普金斯大学动物护理和使用委员会批准的协议进行的，符合ARREAT指南，并按照相关指南和法规进行。
1. 多级
<ol><li>培养基制备
	<ol><li>制备并平衡卵粘液抑制剂，10mg卵粘液抑制剂和每mL厄尔平衡盐溶液（EBSS）10mg白蛋白，使用前30分钟13。在培养箱中与 95% O2：5% CO2 平衡。<ol><li>在此孵育步骤<sup...

<ol>
	<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. 370, (2020).</li><li>Nagalakshmi, U., 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=The+Transcriptional+Landscape+of+the+Yeast+Genome+Defined+by+RNA+Sequencing.">The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing.</a> Science. 320, 1344-1349 (2008).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nature Methods. 5, 621-628 (2008).</li><li>Hwang, B., Lee, J. 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>

MULTI-seq 的强大功能源于来自多个实验条件或模型的数据的无缝集成，以及在成本和限制批次效应方面的巨大优势。利用 MULTI-seq 提供了前所未有的实验室表型深度。非遗传多重检测方法，如细胞散列或细胞核散列，通过使用条形码抗体7，19，20为多路复用样品打开了大门。然而，这依赖于识别细胞或其细胞核上表达的表面蛋白?...

了解基因如何影响细胞命运在询问疾病和胚胎进展等过程中起着关键作用。转录因子与其靶基因之间的复杂关系可以在基因调控网络中分组。越来越多的证据表明，这些基因调控网络处于疾病和进化谱系发展的中心1。虽然以前的技术（如qRT-PCR）专注于单个基因或一组基因，但高通量测序技术的应用允许分析完整的细胞转录组。
RNA-seq提供了对大规模转录组学2，3的一瞥。单细胞RNA测序（scRNA-seq）使研究人员不仅能够分析转录组，还可以将特定细胞类型与基因表达谱联系起来4。这是通过使用已知的基因标记5将单个细胞谱输入到分选算法中来实现的生物信息学。使用脂质标记索引测序（MULTI-seq）进行多重检测，在可收集的scRNA-Seq谱数量方面提供了前所未有的多样性6.这种基于脂质的技术与其他样品索引技术不同，例如依赖于表面抗原和高亲和抗体的存在而不是质膜整合7的细胞散列。现在不仅可以将基因表达谱谱分析为细胞类型，还可以将不同的实验组合到单个测序文库中，从而大大降低了单个scRNA-seq实验的成本6。scRNA-seq的成本对于用于分析许多不同基因型，条件或患者样本的表型实验似乎令人望而却步，但多重检测允许在单个文库中组合多达96个样本6。
通过scRNA-seq分析基因表达并不是唯一一种基于高通量测序的技术，它彻底改变了目前对分子机制如何决定细胞命运的理解。虽然了解细胞中存在哪些基因转录本可以识别细胞类型，但同样重要的是了解基因组组织如何调节发育和疾病进展。早期研究依赖于检测DNase介导的不与组蛋白结合的序列的切割，然后对产生的DNA片段进行测序以鉴定开放染色质的区域。相比之下，转座子可访问染色质测序（scATAC-seq）的单细胞测定允许研究人员用驯化的转座子探测DNA，以便在单核苷酸水平8上轻松谱分析开放染色质。这已经经历了与scRNA-seq类似的缩放，现在研究人员可以识别单个细胞类型并分析数千个单个基因组中的表型8。
scRNA-seq和scATAC-seq的配对使研究人员能够分析数千个细胞，以确定疾病模型和发育过程中的细胞群，基因组组织和基因调控网络9，10，11，12。在这里，作者概述了如何首先利用 MULTI-seq 来凝聚无数动物模型的表型，并采用配对的 scRNA-seq 和 scATAC-seq 来更好地了解这些动物模型中的染色质景观和调控网络。

<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

强大的下一代测序技术提供了强大而全面的分析，以研究视网膜基因调控网络在发育和疾病状态下如何发挥作用。单细胞RNA测序使我们能够在细胞水平上全面分析在视网膜发育和疾病中观察到的基因表达变化，而单细胞ATAC-Seq允许分析染色质可及性和转录因子结合，以相似的分辨率进行分析。这里描述了这些技术在发育中的视网膜中的使用，并演示了MULTI-Seq，其中单个样品用修饰的寡核苷酸 - 脂质复合物标记，使研究人员能够增加单个实验的范围并大大降低成本。

该工作流程列出了使用单细胞测序研究发育表型和调节过程的策略。MULTI-seq 样品多重检测可实现初始低成本表型分析，同时配对采集和固定 scRNA-seq 和 scATAC-seq 样品有助于进行更深入的研究（图 1）。
MULTI-seq 条形码可实现多个样本的组合排序及其随后的计算反卷积。可以根据每个细胞的条形码表达来确定其原产地样品（图2A）。这...

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

在这里，作者展示了MULTI-seq用于表型的效用，以及随后配对的scRNA-seq和scATAC-seq在表征视网膜转录组和染色质可及性图谱方面的效用。

使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析

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

该协议将使使用单细胞RNA-seq进行初始表型分析成为可能。并且，当与单细胞ATAC-seq配对时，基因调控网络的重建。使用MULTI-seq可降低初始研究的成本，并且固定单细胞RNA-seq和单细胞ATAC-seq的配对样品可以对基因调控网络进行时程分析。研究人员可以利用该协议来了解控制发育和疾病进展的遗传网络。首先，向每个样品中加入20微升锚定条形码溶液，然后移液混合。在冰上孵育五分钟。然后向每个样品中加入20微升共锚溶液，并在冰上再孵育五分钟。向每个样品中加入一毫升冰冷的1%BSA和PBS，并在4摄氏度下以300×g离心细胞5分钟。在不干扰细胞沉淀的情况下除去上清液。然后加入400微升冰镇1%BSA和PBS。在 300 x g 下在 4 摄氏度下离心五分钟。使用血细胞计数器确定每个样品的细胞浓度，并计算凝胶珠乳液或GEM所需的细胞总数和体积。将每个样品的计算体积合并到新的无菌1.5毫升管中，并确定合并样品的最终细胞浓度。在GEM生成和条形码后，使用大小选择进行基因表达cDNA纯化，确保不要丢弃第一轮选择中的上清液，其中包含样品条形码。将上清液转移到新的无菌1.5毫升微量离心管中并将其储存在冰上。涡旋基于顺磁珠的尺寸选择试剂，并向上清液中加入260微升试剂和180微升100%异丙醇。将混合物移液 10 次，并在室温下孵育 5 分钟。将试管放在磁性架上，等待溶液清除。溶液澄清后，取出并丢弃上清液。向珠中加入500微升80%乙醇30秒，然后弃去上清液。重复此操作，总共洗涤两次。短暂离心珠子并将它们放回磁性架。将计时器设置为两分钟。然后用P10移液管除去残留的乙醇，并在磁性架上风干珠子剩余的两分钟。从磁性架上取出磁珠，并将其重悬于100微升洗脱缓冲液中。彻底移液以重悬，并在室温下孵育两分钟，然后将磁珠放回磁性架并等待溶液清除。将上清液转移到新鲜的1.5毫升微量离心管中。如图所示重复净化，使用一半体积的洗脱缓冲液。对于用于scATAC序列的样品，使用P200移液管从微量离心管中取出任何液体，并在乙醇干冰中快速冷冻样品。要解离用于scRNA测序的样品中的细胞，请使用木瓜蛋白酶执行解离步骤，如文本手稿中所述。在室温下以300×g离心细胞三分钟，并在不破坏细胞沉淀的情况下除去上清液。加入一毫升冷藏的PBS，混合10次或直到细胞完全悬浮。再次，在4摄氏度下以300×g离心五分钟，然后用PBS重复洗涤两次。开始以最低速度设置涡旋试管，并逐滴加入900微升冷冻甲醇，同时继续轻轻涡旋试管以防止细胞结块。将细胞在冰上孵育15分钟。使用血细胞计数器确定固定细胞的浓度，并使用台盼蓝评估固定步骤的功效。将冷冻组织和固定细胞储存在零下80摄氏度。多序列条形码可实现多个样品的组合测序及其随后的计算反卷积。可以根据每个单元格的条形码表达确定其原产地样本。这些组合样本可以作为单个数据集进行分析，用于细胞聚类和细胞类型鉴定。由于每个细胞在GEM生成之前都有条形码，因此细胞双联体很有可能显示多个MULTI序列条形码的表达，因此可以在聚类和细胞类型鉴定之前识别和去除大多数双峰。单细胞ATAC测序可用于生成具有与单细胞RNA测序发现的细胞类型相匹配的细胞类型的数据集。单细胞RNA测序基因表达和单细胞ATAC测序DNA可及性信息的配对能够重建基因调控网络。尝试实验方案时，请记住，民族醇蒸发的两分钟时间对于后续洗脱步骤中的序列检索至关重要。使用配对的单细胞RNA-seq和单细胞ATAC-seq重建基因调控网络，为基因过表达和敲除研究产生靶标。MULTI-seq可以在此阶段用于鉴定细胞类型特异性效应。

Research

JoVE Journal

Neuroscience

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

脊髓电二：外吸电极的制作

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

科研

神经科学

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

隔离啮齿类动物或人类的鼻嗅干细胞

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

高分辨率实时成像发展中的神经上皮细胞的行为

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

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

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

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

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

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

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

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

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

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

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

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

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

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

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