Name: single-cell rna-seq of defined subsets of retinal ganglion cells
Uploaded: 2017-05-22T12:30:00
Description: Watch this Scientific Journal Video about Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells at JoVE.com

我们要感谢Jennifer Bair和Einat Snir以及爱荷华大学人类遗传学研究所协助准备和处理样品。

所有程序均获得西北大学机构动物护理与使用委员会（IACUC）的批准。 1.电生理解决方案的准备（4小时） <ol><li>通过向999mL反渗透纯化的H 2 O中加入1mL焦碳酸二乙酯（DEPC），使0.1％DEPC处理的H 2 O充分混合，使混合物在室温（RT）下孵育1小时。然后，将DEPC混合的H 2 O在液体循环下高压灭菌15分钟。让DEPC处理的H 2 O在室温下冷却。 </li><li>通过将?...

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我们的协议通过一个快速易用的指南，证明了一种方法，用于准备用于高质量测序的识别形态学类别的单个细胞，对样品几乎没有损伤。在本手稿中，本征感光性视网膜神经节细胞形态学表征，分离并制备用于RNA-Seq。细胞应激可能发生在视网膜处理过程中;因此，在使用不超过4小时之后，我们更换每块纸巾。我们可以通过使用电生理学装置从这些细胞记录并监测其反应来评估细胞的状态，这使得?...

在整个中枢神经系统中观察到神经元多样性，特别是在脊椎动物视网膜中，这是一种由1个神经胶质细胞和6个神经元细胞类型组成的高度专门化的组织，其由视网膜祖细胞1,2,3组产生 。细胞的许多亚型可以在功能上，形态上和遗传上分类。该方案的目标是将细胞类型的遗传变异性与其可识别的功能和/或形态特征相结合。已经鉴定了许多基因用于细胞分类，但是许多亚型仍然没有表征，因为它们代表总体群体的一小部分。这些特定亚型中的基因的鉴定将允许更好地了解视网膜内的神经元多样性，并且还可以揭示其他地方的神经细胞的多样化。福此外，单细胞研究允许揭示新细胞类型，这可能被忽略，因为它们在总体4,5,6,7中的代表性很低。 单细胞转录组学的优点之一是可以发现定义特定细胞亚型的独特标记或标记组合。然后，这些可用于获得对该细胞类型的遗传接触以进行不同的操作。例如，我们正在使用该协议来表征视网膜神经节细胞亚类的细胞类型特异性基因，其表达色素黑素素。荧光标记物在表达黑色素的视网膜神经节细胞中的使用使得能够研究这些细胞，因为它们由于其已知基因的表达而聚集在一起。有趣的是，这种细胞群有五种已知的亚型老鼠视网膜中的信号8 。因此，为了从每种类型的细胞中分离RNA，我们在转基因模型中已经使用确定的形态学分类，以在细胞分离之前鉴定每种亚型。这种技术允许表征细胞以及直接从视网膜分离，而不需要组织解离，这可能导致细胞内的应激反应和由于切断的树突引起的污染9 。 随着RNA-Seq方法的不断发展，过去几年中已经有了许多新技术。这些工具允许最大化的细胞获取和更高的成本效率，同时接近手头4，7，10，11，12，13的问题。但是，虽然这些技术一直是优秀的踏脚石，还有一些障碍仍然遇到这个协议能够解决。首先，许多目前的程序将细胞与解离的组织分离，并尝试使用主成分分析或事后分层聚类来确定细胞分类。依靠这些工具来分类亚型可能不会产生可靠的结果，并且可能迫使人们找到新的方法来验证这一数据，以便将遗传标记与功能性细胞类型相关联。其他方案中解离的要求有时会导致组织损伤，并可能导致神经元过程被切断，导致mRNA潜在的丧失。此外，在解离的细胞制剂中，应激反应可能开始影响这些细胞的转录组14 。该协议通过确定隔离前的功能细胞类型来克服这些挑战，并且更好地维持h通过保持视网膜组织完整而保持细胞。 2014年引入了一种技术，其中包括活细胞转录组的体内分析。虽然这种技术允许以对组织的最小的机械破坏来检查转录组，但是在检查其转录组之前没有使用非常特异性的记录小鼠，其缺乏对组织内的特定细胞类型进行分类的能力。我们的方案不需要特定的记者，因为我们利用细胞填充和电生理学在细胞分离前表征细胞。此前协议的另一个限制是需要特定的波长来激发可光活化的元件，而我们的方案允许使用荧光报告物和荧光染料，这些荧光染料可以容易地获得，也可以由每个实验室单独选择。然而，其他实验室却结婚了两种电话生物学和转录组学研究细胞多样性。已经在分离的神经元16上进行使用膜片钳记录来表征细胞在其分离之前的功能，并且在某些情况下，已经使用微阵列分析17进行这些研究。这些方法遇到相同的并发症，因为它们需要组织解离或使用微阵列技术，其依赖于样品与可用探针的杂交。最近的进展之一是Patch-Seq的发展，Patch-Seq是一种结合使用膜片钳记录和RNA-Seq技术来了解全脑切片细胞的技术。虽然这种技术与本文提出的方案有相似之处，但重要的是要注意，我们的方法允许组织保持完整，以保持细胞的健康。在这里，我们提出了一个优化协议该联盟产生高质量的单细胞文库以使用RNA-Seq获得高读取深度和测绘覆盖率。

<table border="0" cellpadding="0" cellspacing="0" width="907">
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		<tr height="20">
			<td height="20" style="height:20px;width:257px;">Ames&#39; Medium</td>
			<td style="width:171px;">Sigma Aldrich</td>
			<td style="width:108px;">A1420-10X1L</td>
			<td style="width:372px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">K-gluconate</td>
			<td>Spectrum Chemical</td>
			<td>PO178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma Aldrich</td>
			<td>D5758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 594 Hydrazide</td>
			<td>Invitrogen</td>
			<td>A10442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS005273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyaluronidase</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS002592</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish (35mm diameter)</td>
			<td>Thermo Fisher Scientific</td>
			<td>153066</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ophthalmologic scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">#5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microplate Shaker</td>
			<td>Fisher Scientific</td>
			<td>13-687-708</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Micropipette</td>
			<td>Sutter</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micropipette Puller</td>
			<td>Sutter</td>
			<td></td>
			<td>P-1000 horizontal pipette puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1mL syringe</td>
			<td>Fisher Scientific</td>
			<td>14-823-2F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Flexible tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCL lysis buffer</td>
			<td>Qiagen</td>
			<td>1031576</td>
			<td>Lysis Buffer 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free Water</td>
			<td>Qiagen</td>
			<td>129112</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2 ml PCR tubes</td>
			<td>Eppendorf</td>
			<td>30124359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl Alcohol, Pure</td>
			<td>Sigma Aldrich</td>
			<td>E7023</td>
			<td>Ethanol</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analog Vortex Mixer</td>
			<td>Thermo Fisher Scientific</td>
			<td>02215365</td>
			<td>Vortex</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mini Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-090-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt RNAClean XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63987</td>
			<td>RNA magnetic beads</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MagnaBlot II Magnetic Separator</td>
			<td>Promega</td>
			<td>V8351</td>
			<td>Magnetic stand</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml MCT Graduated Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Smart-Seq v4 Ultra Low Input RNA Kit</td>
			<td>Clontech</td>
			<td>634888</td>
			<td>Reagents for Reverse Transcription and PCR Amplification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Lysis Buffer</td>
			<td></td>
			<td></td>
			<td>Lysis Buffer 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5X Ultra Low First-Strand Buffer</td>
			<td></td>
			<td></td>
			<td>Buffer 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3&#39; SMART-Seq CDS Primer II A</td>
			<td></td>
			<td></td>
			<td>Primer II</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SMART-Seq v4 Oligonucleotide</td>
			<td></td>
			<td></td>
			<td>Oligonucleotide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SMARTScribe Rverse Transcriptase</td>
			<td></td>
			<td></td>
			<td>Reverse Transcriptase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2X SeqAmp PCR Buffer</td>
			<td></td>
			<td></td>
			<td>PCR Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Primer II A</td>
			<td></td>
			<td></td>
			<td>PCR Primer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SeqAmp DNA Polymerase</td>
			<td></td>
			<td></td>
			<td>DNA Polymerase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mastercycler pro S</td>
			<td>Eppendorf</td>
			<td>950030020</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>DNA magnetic beads</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HS Bioanalyzer Chips &#38; Reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit HS Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32851</td>
			<td>For the calculation of sample concentrations</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit Assay Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit 2.0 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT DNA Sample Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>Reagents for Tagmentation and Index Coupling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TD Buffer</td>
			<td></td>
			<td></td>
			<td>Buffer 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATM</td>
			<td></td>
			<td></td>
			<td>Tagmentation Mix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NT Buffer</td>
			<td></td>
			<td></td>
			<td>Tagmentation Neutralizing Buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NPM</td>
			<td></td>
			<td></td>
			<td>PCR Master Mix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td>Indices for Tagmentation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N501</td>
			<td></td>
			<td></td>
			<td>White 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N502</td>
			<td></td>
			<td></td>
			<td>White 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N701</td>
			<td></td>
			<td></td>
			<td>Orange 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N702</td>
			<td></td>
			<td></td>
			<td>Orange 2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiSeq 2500</td>
			<td>Illumina</td>
			<td>SY-401-2501</td>
			<td>For completing sequencing of samples</td>
		</tr>
	</tbody>
</table>

single-cell rna-seq of defined subsets of retinal ganglion cells

细胞类型特异性标记的发现可以提供对细胞功能的了解和细胞异质性的起源。随着对神经元多样性的更好理解的推动，识别表达定义各种细胞亚群的基因是重要的。视网膜作为中枢神经系统多样性研究的优秀模式，由多种主要细胞类型组成。每个主要类别细胞的研究已经产生了便于识别这些群体的遗传标记。然而，在这些主要视网膜细胞类别的每一个中存在多种细胞亚型，并且这些亚型中很少有已知的遗传标记，尽管许多已经通过形态或功能表征。对于个别视网膜亚型的遗传标记的知识将允许与特定视觉功能相关的脑靶标的研究和绘图，并且还可以洞察基因网络维持细胞多样性。用于鉴定亚型遗传标记的现有途径具有缺点，例如测序后细胞类型的分类。这为数据分析提出了挑战，需要严格的验证方法来确保集群包含相同功能的单元格。我们提出了一种用于在分离和测序之前鉴定细胞的形态和功能的技术，这将允许更容易鉴定亚型特异性标记。这种技术可以扩展到非神经元细胞类型，以及稀有的具有微小变异的细胞群体。该协议产生优质的数据，因为许多库为单个单元提供了大于2000万次读取的读取深度。该方法克服了单细胞RNA-Seq提出的许多障碍，并且可能适用于以直接和高效的方式分析细胞类型的研究人员。

细胞类型在染料注射后容易分类 图1显示荧光示踪剂填充之前和之后的GFP + RGC的实例。基于其在转基因品系中GFP的表达鉴定该细胞（ 图1A ）。在细胞的细胞上形成细小的拉丝玻璃电极的密封。为了表征亚型，将荧光染料注入到血浆中并允许填充所有相关的过程（ 图1B ）。通过观察，该细胞?...

Watch this Scientific Journal Video about Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells at JoVE.com

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

在这里，我们提出了一种组合方法，用于在分离之前对神经元细胞类型进行分类，以及随后对单细胞转录组进行表征。该协议优化了用于成功的RNA测序（RNA-Seq）的样品的制备，并描述了专门为增强细胞多样性的理解而设计的方法。

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Research

JoVE Journal

Neuroscience

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

科研

神经科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Single-cell Profiling of Developing and Mature Retinal Neurons

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

Viral Tracing of Genetically Defined Neural Circuitry

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...