Name: single-cell rna sequencing of fluorescently labeled mouse neurons using manual sorting and double in vitro transcription with absolute counts sequencing (diva-seq)
Uploaded: 2018-10-26T15:00:00
Description: Watch this Scientific Journal Video about Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-

这项工作得到了 NIH (5R01MH094705-04 和 R01MH109665-01 至 Z.J.H.)、CSHL 罗伯逊神经科学基金 (Z.J.H.) 和 NARSAD 博士后奖学金 (美联社) 的资助。

所有的程序, 包括动物科目已经批准了 IACUC 在寒冷的春天海港实验室, 纽约州 (IACUC #16 13-09-8)。
1. 荧光标记的小鼠神经元的手动排序
<ol><li>拉玻璃显微操作针 (见材料表) 到10–15µm 出口直径使用毛细管拉拔器与以下设置: 热: 508, 拉: 空白, 上: 空白, 时间: 空白。</li>
	<li>使用合适的油管接头将 120–150 cm 柔性硅胶管 (直径约0.8 毫米) 添加到0.2 µm 聚偏二氟化 (PVDF) 膜?...

<ol>
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手动排序协议适用于神经元种群的受监督 RNA 序列, 它们要么是稀疏标记在小鼠大脑中, 要么代表一个罕见的细胞种群, 否则使用当前高通量细胞进行研究是不可行的。排序和放大方法。根据喷嘴尺寸和所需的事件速率, 在 9–14 psi 范围内, 受流管的细胞通常会承受鞘和采样线压力。此外, 当从喷嘴中弹出时, 细胞可以在收集管表面坚硬, 或涂有样品缓冲液的油井, 造成冲击应力。在手动分拣过程中, 这...

单细胞 rna 测序 (rna 序列) 改变了转录组研究。虽然可以使用各种技术 (如水滴1、2、微流体3、nanogrids4和 microwells5) 执行大型单细胞 RNAseq, 但大多数方法无法对定义的像元类型进行排序表达基因编码的荧光基团为了隔离选择的细胞种群, 荧光活化细胞分类 (流式) 通常用于在单细胞模式下对标记的细胞进行排序。然而, 流流仪有一些限制, 需要细致的样品处理步骤。首先, 通常需要大量的输入单元格 (通常每毫升有数个百万细胞), 其中包含标记种群的显著分数 (> 15–20%)。其次, 细胞制剂可能需要多轮的密度梯度离心步骤, 以除去可能堵塞喷嘴或流动细胞的胶质碎片、碎屑和细胞团簇。第三, 流式细胞仪通常采用染色和脱色步骤进行活/死染色 (例如4′-、6-diamidino-2-苯基吲哚 (DAPI)、丙啶碘化物 (PI) 和 Cytotracker 染料), 这会占用更多时间。第四, 作为双色排序的经验法则 (如 DAPI 和绿/红荧光蛋白 (GFP/RFP)), 通常需要两个样本和一个控制, 要求除了所需的鼠标应变之外, 还需要处理未标记的样本。第五, 过滤通常在样品排序之前和期间多次执行, 以主动防止流式机中的采样线堵塞。第六, 必须在最常用的流式处理装置中分配时间, 以初始化和稳定流体流动并执行液滴校准。第七, 控制样本通常在实际样本采集之前按顺序运行, 以设置补偿矩阵、双峰抑制、设置门等.用户可以提前执行步骤六和七, 或者要求技术人员并行协助。最后, 后流后, 经常有步骤, 以确保只有标记的单个单元格中存在每个井;例如, 通过在高内容筛选设置 (如快速平板成像仪) 中检查样本。
为了规避上述步骤, 并促进相对快速、有针对性的单荧光标记神经元的小种群序列, 我们描述了一个手动分拣程序, 后跟两轮高度敏感的体外扩增协议, 称为双体外转录与绝对计数排序 (天后-Seq)。RNA 扩增和 cDNA 文库的生成是从 Eberwine等。6和 Hashimshony等。7、有一定的修改, 适合小鼠中间神经元的蜂窝体积较小;此外, 我们还发现, 它对兴奋性锥体神经元同样有用。

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">ERCC RNA Spike-In Control Mixes</td>
			<td style="width:123px;">Thermo Fisher</td>
			<td style="width:344px;">Cat# 4456740</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">SuperScript III</td>
			<td>Thermo Fisher</td>
			<td>Cat# 18080093</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">RNaseOUT Recombinant Ribonuclease Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>Cat# 10777019</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">RNA fragmentation buffer</td>
			<td>New England Biolabs</td>
			<td>Cat# E6105S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">RNA MinElute kit</td>
			<td>Qiagen</td>
			<td>Cat# 74204</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Antarctic phosphatase</td>
			<td>New England Biolabs</td>
			<td>Cat# M0289</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Poly nucleotide kinase</td>
			<td>New England Biolabs</td>
			<td>Cat# M0201</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">T4 RNA ligase2, truncated</td>
			<td>New England Biolabs</td>
			<td>Cat# M0242</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Ampure Xp magnetic&#160; beads</td>
			<td>Beckman Coulter</td>
			<td>Cat# A63880</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">SPRIselect size selection magnetic beads</td>
			<td>Thermo Fisher</td>
			<td>Cat# B23317</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">DL-AP5</td>
			<td>Tocris</td>
			<td>Cat# 0105</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">CNQX</td>
			<td>Tocris</td>
			<td>Cat# 1045</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">TTX</td>
			<td>Tocris</td>
			<td>Cat# 1078</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Protease from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# P5147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Message Amp II kit</td>
			<td>Thermo Fisher</td>
			<td>Cat# AM1751</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Carbogen</td>
			<td>Airgas</td>
			<td>Cat# UN3156</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Sylgard 184</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# 761036</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Illumina TrueSeq smallRNA kit</td>
			<td>Illumina</td>
			<td>Cat# RS-200-0012</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Bioanalyzer RNA Pico chip</td>
			<td>Agilent</td>
			<td>Cat# 5067-1513</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Bioanalyzer High Sensitvity DNA chip</td>
			<td>Agilent</td>
			<td>Cat# 5067-4626</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:261px;">Dissection microscope with fluorescence and bright field illumination with DIC optics.&#160; (Leica model MZ-16F).</td>
			<td>Leica</td>
			<td>Model MZ-16F</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:261px;">Glass microcapillary: Borosilicate capillary tubes 500/pk. OD= 1 mm, ID=0.58 mm, wall= 0.21 mm, Length= 150 mm.</td>
			<td>Warner instruments</td>
			<td>Model GC100-15, Order# 30-0017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Capillary pipette puller</td>
			<td>Sutter Instruments Co</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Vibratome</td>
			<td style="width:123px;">Thermo Microm</td>
			<td>HM 650V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome tissue cooling unit</td>
			<td>Thermo Microm</td>
			<td>CU 65</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell rna sequencing of fluorescently labeled mouse neurons using manual sorting and double in vitro transcription with absolute counts sequencing (diva-seq)

单细胞 rna 测序 (rna 序列) 现在是一个广泛实施的工具, 用于测定基因表达。商业上可用的单细胞 RNA 测序平台不加区别地处理所有输入单元。有时, 在上游使用荧光活化细胞分类 (流化) 来隔离特定标记的感兴趣种群。流化的局限性是需要大量的输入细胞具有显著标记的分数, 这是不切实际的收集和分析稀有或稀疏标记的神经元种群从小鼠大脑。在这里, 我们描述了一种手动收集稀疏荧光标记单个神经元从新鲜分离的小鼠脑组织的方法。此过程允许捕获具有高纯度的单标记神经元, 并随后与体外转录放大协议相结合, 以保留内源转录比。我们描述了一种双线性放大方法, 它使用唯一的分子标识符 (医药学) 来生成单个 mRNA 计数。两轮的扩增会导致每个单细胞的基因检测高度。

使用上面描述的协议, gaba 能神经元被手动排序 (图 1) 和 RNA 被放大, 然后被制成 cDNA 文库 (图 2) 和排序在高深度8。放大的 RNA (aRNA) 产品介于 200–4,000 bp 的大小之间, 峰值分布略高于 500 bp (图 3A)。珠纯化 cDNA 文库进一步大小-限制由第二轮纯化使用珠子, 消除小碎片小于 200 bp (<strong class="xfi...

Watch this Scientific Journal Video about Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

荧光标记小鼠神经元的单细胞 RNA 序列的人工分选和双体外转录与绝对计数测序 (天后-Seq)

本协议描述了将单个荧光标记神经元与体外转录的 mRNA 扩增分离以进行高深度单细胞 RNA 测序的人工分拣程序。

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

该方法有助于解决单神经元分离和测序领域的主要障碍。例如从用户定义的脑区域收集特定人群或荧光标记神经元的亚组。该技术的主要优点是，它确保捕获感兴趣的单个神经元而不污染碎片。它也相对温和，这对于脆弱的神经元类型来说很重要，当受到传统 FACS 分拣的流体压力时，这些神经元类型会迅速死亡。首先使用毛细管拉拔器制备直径为 10 至 15 微米的拉玻璃微胶囊。使用细剪刀切割毛细管的尖端，以获得所需的板直径。然后使用管连接器将一个 120 至 150 厘米长的柔性硅胶管连接到 0.2 微米 PVDF 膜注射器过滤器和双向管阀，内径为 0.8 毫米。通过气石冒泡 5%二氧化碳平衡氧 15 分钟或直到溶液完全清除，准备 500 毫升的冷冻人工脑脊髓液或 ACSF 和氧合。准备三个 150 毫升的烧杯，每个装有 100 毫升的 ACSF。一是加入一种活动阻滞剂的鸡尾酒。另一方面，将蛋白酶的链球菌分数添加到每毫升一毫克的最终浓度中。到最终烧杯，将FBS加入到1%的最终浓度中，通过气石加入5%的二氧化碳平衡氧氧。最后，制作三个长茎巴斯特移液器，通过滚动在明火上，减少出口直径。用小剪刀打开颅骨，用细钳拔出新鲜大脑，而不损害皮层，从安乐死小鼠身上解剖大脑。将大脑放在冰冷和含氧的ACSF中，在整个分节期间，留下一个气石附着在5%的二氧化碳平衡氧上。将大脑放在振动夹头上，在300微米厚度下切割日冕或下垂部分。根据需要收集尽可能多的部分，以获得至少 10 到 50 个标记单元格。将切片放在放在烧杯内的棉网切片架上，以便它们沐浴在含氧 ACSF 中。将切片用活性阻滞剂将切片移入含有 ACSF 的烧杯中，并在室温下块块 15 至 20 分钟，同时使用 Airstone 冒泡氧气。使用蛋白酶溶液将切片移动到含有 ACSF 的烧杯中，在室温下进行轻度消化。消化后，将切片用活性阻滞剂溶液将切片移动回含有ACSF的烧杯中洗净蛋白酶，在室温下5至10分钟。继续冒泡氧气。接下来，在室温下准备一个含有ACSF的100毫米培养皿，并在室温下将各个部分移动到磁盘中进行微截。在荧光解剖范围内，使用一对细钳子对具有至少10至50个细胞的微解剖区域和感兴趣层。使用巴斯德移液器，将微解剖件移动到包含约 0.8 毫升 1%FBS 的 2 毫升微离心管中 ACSF 溶液中。在室温下对微烟管中的解剖组织进行滴定。每个火焰巴斯德移液器以最大开始，以最小的出口直径结束，执行大约 10 次冲程。将分离的细胞分配成含有含氧ACSF的100毫米培养皿，等待5至7分钟，让细胞逐渐沉淀。在解剖显微镜下观察 GFP 和 RFP 信号。通过检查亮场照明下的形态来识别碎片。一旦发现有小碎片的细胞区域，使用毛细管并附着在管子上，用舌头阻塞管阀的端接细胞。然后将毛细管靠近细胞。松开毛细管上的块，然后快速再次阻塞，使用毛细管作用捕获细胞。将毛细管移动到 100 毫米培养皿中，配以新鲜含氧 ACSF，轻轻吹入管中，同时观察荧光下毛细管尖端，将细胞放入培养皿中。重复收集过程，直到收集 100 到 150 个电池，确保在亮场差分干涉对比度光学中污染物（如碎屑）最小。最后，每次使用新鲜的微毛细管移液器，选择一个细胞，并在不超过0.5微升中将它排出0.2微升微烟管，其中包含一个微升的样品收集缓冲液和底像。断开微烟管中的移液器尖端，以确保细胞留在收集缓冲器中。将管子放在干冰上，冷冻细胞。存放在零下80摄氏度的冰柜中，直到处理RNA扩增。GABAergic神经元从小鼠大脑的银骨皮层分离。从人工排序的神经元中放大的RNA大小在200到4000个碱基对之间，峰值分布略高于500个碱基对。珠子纯化后，cDNA库被第二轮纯化所限制，使用珠子消除小于200个碱基对的碎片，并在大约350个基对的峰值。排序显示每个单元格的平均映射读取数为 4.8 倍 10 至第 5 中位数，或 6.9 倍 10 到 5 的平均映射读取。使用 UMI 重复去除 RNA 后，每个单个细胞的 1.0 倍 10 到第 5 中位数，或 1.4 倍 10 到 5 的平均唯一读取每个细胞。在每个单细胞外部RNA控制联合体中，RNA的峰值被用作内部控制，以计算添加到样品中的分子的绝对数量。有输入与观测计数的线性关系，斜率为 0.92，调整后的 R 平方为 0.94。使用这个程序，每个单个神经元平均检测出大约1万个基因，超过95%的单细胞检测出6000多个基因。该技术开发后，为分子生物学家探索不同特征的小鼠皮质内维龙的转录组铺平了道路，并确定了负责编码细胞特性的精选基因类别。

Research

JoVE Journal

Neuroscience

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

科研

神经科学

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

在使用单细胞RT - PCR技术的黑质神经元电压门控钾通道mRNA表达分析

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

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 ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

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

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 ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFRÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â±+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis

低细胞染色质免疫沉淀测序分析 Olig2 基因组结合位点在急性纯化 PDGFRα + 细胞中的转录因子鉴定

In mammalian cells, gene transcription is regulated in a cell type specific manner by the interactions of transcriptional factors with genomic DNA. ...

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

膜片钳后单细胞多重反转录聚合酶链反应

The cerebral cortex is composed of numerous cell types exhibiting various morphological, physiological, and molecular features. This diversity hampers ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...