Name: isolation and transcriptome analysis of plant cell types
Uploaded: 2023-04-07T12:40:00
Description: Watch this Scientific Journal Video about Isolation and Transcriptome Analysis of Plant Cell Types at JoVE.com

我们在上海交通大学农业与生物学院的单细胞多组学设施中建立了该方案，并得到了国家自然科学基金（批准号32070608），上海浦江计划（批准号20PJ1405800）和上海交通大学（批准号Agri-X20200202，2019TPB05）的支持。

以下方案已针对拟南芥野生型（WT）种子进行了优化，没有荧光和以下根细胞类型的荧光标记系：木质部 - 极周周期细胞（J0121），侧根初始细胞，侧根帽细胞（J3411），内胚层和皮质细胞（J0571）（图1A）。所有标记系均来自商业来源（见材料表），但侧根起始细胞标记系除外，该标记系是通过在先前发表的报告14之后将GATA23启动子驱动?...

<ol>
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基于Smart-seq2的方案可以从数百个细胞中生成可靠的测序文库8。起始材料的质量对于转录组分析的准确性至关重要。FACS是制备目标细胞的强大工具，但该过程，尤其是原生增殖步骤，必须针对植物应用进行优化。激光捕获显微切割（LCM）或手动解剖细胞也可以用作输入25，26，因此此处提供的协议可以潜在地用于具有或不具有已知标记?...

细胞是所有生物体的基本单位，执行结构和生理功能。尽管多细胞生物中的细胞显示出明显的同步性，但不同类型和单个细胞的细胞在发育和环境反应过程中的转录组存在差异。高通量单细胞RNA测序（scRNA-seq）为了解细胞异质性提供了前所未有的能力。在植物科学中应用scRNA-seq有助于成功构建植物细胞图谱1，已被用于鉴定植物组织中的稀有细胞分类群2，提供了对植物组织中细胞类型组成的见解，并已用于鉴定细胞身份和植物发育和分化过程中使用的重要功能。此外，还可以推断植物组织1，2，3的时空发育轨迹，以发现新的标记基因4并使用scRNA-seq研究重要转录因子5的功能，以揭示不同植物中相同细胞类型的进化保守性3.非生物胁迫是影响植物生长发育的最重要环境影响之一。通过单细胞转录组测序探索不同处理条件下植物组织中细胞类型组成的变化，还可以解决非生物胁迫响应机制6。
使用scRNA测序解决细胞类型之间的转录异质性的潜力取决于细胞分离方法和测序平台。荧光激活细胞分选（FACS）是一种广泛使用的技术，用于根据光散射和细胞的荧光特性分离细胞亚群以获得scRNA-seq。转基因技术开发荧光标记系，大大提高了FACS7细胞分离的效率。使用Smart-seq28 进行scRNA-seq进一步增强了解剖细胞异质性的能力。Smart-seq2方法具有良好的基因检测灵敏度，即使在低转录本输入的情况下也能检测基因9。除了批量细胞类型采集外，现代细胞分选仪还提供单细胞索引分选格式，允许使用 Smart-seq210 或其他多重 RNA-seq 方法（如 CEL-seq211）以单细胞分辨率进行转录组分析。单细胞或细胞类型分选可用于许多其他下游应用，例如平行多组学研究12，13。这里介绍的是一种强大且通用的方案，用于通过 FACS 从 拟南芥 标记细胞系的根中分离植物细胞类型，例如木质部极周周期细胞、侧根帽细胞、侧根初始细胞、皮质细胞和内胚层细胞。该协议还涉及构建用于下游转录组分析的Smart-seq2文库。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m strainer</td>
			<td>Sorfa&#160;</td>
			<td>622110</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Yeasen</td>
			<td>70101ES76</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent fragment analyzer</td>
			<td>Aglient</td>
			<td>Aglient 5200</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent high-sensitivity DNA kit</td>
			<td>Aglient</td>
			<td>DNF-474-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampure XP beads</td>
			<td>BECKMAN</td>
			<td>A63881</td>
			<td></td>
		</tr>
		<tr>
			<td>Betaine</td>
			<td>yuanye</td>
			<td>S18046-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Mr Muscle</td>
			<td>FnBn83BK</td>
			<td>20% (v/v) bleach</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>sigma</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>yuanye</td>
			<td>S24109-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase R10</td>
			<td>Yakult (Japan)</td>
			<td>9012-54-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase RS</td>
			<td>Yakult (Japan)</td>
			<td>9012-54-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube (1.5 mL)</td>
			<td>Eppendolf</td>
			<td>30121589</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase, RNase, DNA and RNA Away Surface Decontaminants</td>
			<td>Beyotime</td>
			<td>R0127</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (10 mM)</td>
			<td>NEB</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (0.1 M)</td>
			<td> 
			invitrogen</td>
			<td>18090050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>100092680</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>BD FACS Melody</td>
			<td>BD-65745</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>Sony</td>
			<td>SH800S</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip&#160; (1000 &#181;L)</td>
			<td>Thermo Scientific</td>
			<td>TF112-1000-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip&#160; (200 &#181;L)</td>
			<td>Thermo Scientific</td>
			<td>TF140-200-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip (10 &#181;L)</td>
			<td>Thermo Scientific</td>
			<td>TF104-10-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip (100 &#181;L)</td>
			<td>Thermo Scientific</td>
			<td>TF113-100-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ni-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Four-Dimensional Rotating Mixer</td>
			<td>Kylin -Bell</td>
			<td>BE-1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemicellulase</td>
			<td>sigma</td>
			<td>9025-56-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IS PCR primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-AAGCAGTGGTATCAACGCAGAG 
			T-3&#39;</td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix(2X)</td>
			<td>Roche&#160;</td>
			<td>7958935001</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Macerozyme R10</td>
			<td>Yakult (Japan)</td>
			<td>9032-75-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic separation stand</td>
			<td>invitrogen</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>aladdin</td>
			<td>69-65-8</td>
			<td></td>
		</tr>
		<tr>
			<td>MES</td>
			<td>aladdin</td>
			<td>145224948</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#160;</td>
			<td>yuanye</td>
			<td>R21455-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuges</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5425</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-mini-centrifuge</td>
			<td>Titan</td>
			<td>Timi-10k</td>
			<td></td>
		</tr>
		<tr>
			<td>MS</td>
			<td>Phytotech</td>
			<td>M519</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit</td>
			<td>illumina</td>
			<td>FC-131-1024</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo-dT30VN primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-AAGCAGTGGTATCAACGCAGAG 
			TACTTTTTTTTTTTTTTTTTTTTTTT 
			TTTTTTTTTTVN-3&#39;</td>
		</tr>
		<tr>
			<td>PCR instrument</td>
			<td>Thermal cycler</td>
			<td>A24811</td>
			<td></td>
		</tr>
		<tr>
			<td>Pectolyase</td>
			<td>Yakult (Japan)</td>
			<td>9033-35-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Plant marker lines</td>
			<td>Nottingham Arabidopsis Stock Centre (NASC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 1x dsDNA HS Assay Kit</td>
			<td>invitrogen</td>
			<td>Q33231</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer</td>
			<td>invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor&#160;</td>
			<td>Thermo Scientific</td>
			<td>EO0382</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>invitrogen</td>
			<td>10977023</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution A</td>
			<td></td>
			<td></td>
			<td>400 mM mannitol, 0.05 % BSA , 20 mM MES (pH5.7), 10 mM CaCl2, 20 mM KCl</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
			<td></td>
			<td>1 % (w/v)cellulase R10, 1 % (w/v) cellulase RS, 1 %&#160; (w/v)hemicellulase, 0.5 %&#160; (w/v)pectolyase and 1 %&#160; (w/v) Macerozyme R10 of in a fresh aliquot of solution A</td>
		</tr>
		<tr>
			<td>Sterile pestle</td>
			<td>BIOTREAT</td>
			<td>453463</td>
			<td></td>
		</tr>
		<tr>
			<td>Strainer (40 &#181;m )</td>
			<td>Sorfa&#160;</td>
			<td>251100</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript enzyme (200 U/&#181;L)</td>
			<td>invitrogen</td>
			<td>18090050</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript VI buffer (5x)</td>
			<td>invitrogen</td>
			<td>18090050</td>
			<td></td>
		</tr>
		<tr>
			<td>T0est tube (5 mL)</td>
			<td>BD Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-walled PCR tubes with caps (0.5 mL)</td>
			<td>AXYGEN</td>
			<td>PCR-05-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon Biotech</td>
			<td>A600198-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>TSO primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-AAGCAGTGGTATCAACGCAGAG 
			TACATrGrG+G-3&#39;</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Titan</td>
			<td>VM-T2</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and transcriptome analysis of plant cell types

在多细胞生物中，发育编程和环境反应在不同的细胞类型甚至细胞内可能高度不同，这被称为细胞异质性。近年来，单细胞和细胞类型分离与二代测序（NGS）技术相结合已成为研究单细胞分辨率生物过程的重要工具。然而，由于植物细胞壁的存在，分离植物细胞相对困难，这限制了单细胞方法在植物中的应用。该协议描述了基于荧光激活细胞分选（FACS）的单细胞和细胞类型与植物细胞分离的稳健程序，适用于下游多组学分析和其他研究。使用 拟南芥 根荧光标记系，我们展示了如何分离特定细胞类型，例如木质部 - 极周周期细胞，侧根初始细胞，侧根帽细胞，皮质细胞和内胚层细胞。此外，还提供了使用Smart-seq2的有效下游转录组分析方法。细胞分离方法和转录组分析技术可以适应其他细胞类型和植物物种，在植物科学中具有广泛的应用潜力。

原生质体分离 该协议对于荧光 拟南芥 根标记系的原生质体分选有效。这些标记系是通过荧光蛋白与靶细胞类型中特异性表达的基因融合或使用增强子捕获系开发的（图1）。许多组织和器官已被解剖成在模式植物和作物中表达特定荧光标记物的细胞类型。
FACS 群体、分选细胞和文库质量控制 通过使用野生型植物作为...

Watch this Scientific Journal Video about Isolation and Transcriptome Analysis of Plant Cell Types at JoVE.com

Isolation and Transcriptome Analysis of Plant Cell Types

高通量scRNA-seq方法的可行性和有效性预示着植物研究的单细胞时代。这里介绍的是一个强大而完整的程序，用于分离特定的拟 南芥 根细胞类型以及随后的转录组文库构建和分析。

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

对单细胞或细胞类型的转录组分析可以检测到基因表达被异质性掩盖的细微差异，这是揭示复杂的细胞内调节机制的有力工具。荧光活化细胞分选后进行RN-seq分析可以在单细胞或大细胞类型模式下进行，具有高转录本检测灵敏度并与其他多组学方法兼容。首次使用该方案的用户需要注意原生质体分选和RNA-seq文库制备中的一些步骤。与张军一起，我们实验室的博士后谢荣荣博士将帮助演示该程序。首先，将拟南芥野生型和荧光标记系种子放入20%漂白剂中，并使用室温旋转培养箱孵育15分钟以对种子进行灭菌。在无菌工作台上工作时，用双蒸水冲洗种子三到五次。将野生型和报告系种子铺在半强度MS培养基上，每体积琼脂重量为0.8%。在四摄氏度下对植物分层两天。然后在 23 小时光照和 18 小时黑暗循环下在 23 摄氏度下垂直种植五天。在实验前，在冰上轻轻解冻称为溶液A和溶液B的原生质持久溶液。用干净的刀片或剪刀从植物上剪掉根部，并将根切成大约 0.5 厘米的碎片。将切碎的根浸入1.5毫升溶液B中，然后在室温下轻轻旋转1.5至2小时。通过40微米过滤器网过滤所得的根原生质体，并用一至两毫升溶液A冲洗网状物.将合并的滤液在300G下在4摄氏度下离心五分钟。使用移液管弃去上清液，将细胞沉淀重悬于500至600微升溶液A中，并立即将其置于冰上。对于细胞分选，将重悬的细胞溶液转移到新的五毫升试管中。填充护套液罐并检查废物罐。然后打开荧光激活细胞分选或FACS机器。在分拣机上执行仪器设置和自动校准步骤。选择荧光通道并使用没有荧光的野生型植物作为基线对照。根据荧光强度和前向散射和侧向散射单体调整分拣门。将500微升溶液A加入1.5毫升收集管后，开始分选，每管收集2， 000至3， 000个细胞。分拣后，立即将样品放在冰上。将含有细胞的收集管在300G和4摄氏度下离心5分钟，并用移液管除去上清液。取两微升分选的细胞，并使用荧光显微镜检查荧光。如果不立即使用，请将分选的细胞储存在零下 80 摄氏度。对于单细胞指数分选，将带膜的 96 孔板放入适配器中。校准板的位置，使液滴落在板的中心孔中。从 96 孔板上取下膜并将 96 孔板放入适配器中。排序时选择单细胞排序模式。将目标数量的排序单元格输入为一个，然后开始排序。在开始实验之前，用表面净化剂和75%乙醇清洁工作台。仅将所有设备用于测序文库制备。将一微升新鲜制备的混合物A加入分选样品后，用无菌研杵研磨。使用游离RNAse的水，将体积补足至14微升。然后将每个样品转移到0.2毫升薄壁PCR管中。然后制备混合物B.向每个含有样品的PCR管中加入4.4微升混合物B，并通过轻轻移液混合。将样品在 72 摄氏度下孵育三分钟。孵育后，立即将样品放在冰上，将寡核苷酸dT杂交到poly-A尾部。将逆转录反应混合物或混合物C在21.6微升中制备到每个含有样品的试管中。根据逆转录程序，在普通PCR仪器中对样品进行逆转录反应。将40.8微升新鲜制备的预扩增反应混合物或混合物D加入逆转录反应产物中，并运行预扩增程序。当反应刚刚进入指数阶段时，可以通过定量PCR确定扩增周期。要纯化预扩增反应产物，请将预扩增产物从PCR管转移到1.5毫升管中。向每个样品中加入 48 微升 AMPure XP 磁珠，并在孵育前通过移液轻轻混合。将含有样品和珠子的 1.5 毫升管放在磁分离台上五分钟。小心地从样品中丢弃上清液，不要干扰珠子。将它们重悬于200微升80%乙醇中后，将它们放在磁分离架上再放置三分钟。丢弃含有上清液的乙醇后，将样品在管中风干10分钟。将磁珠重悬于20微升无核酸酶的水中，并在室温下孵育五分钟。然后将它们放在磁分离架上五分钟。使用移液管，将每个管中的18微升上清液转移到新的1.5毫升离心管中。最后，按照文中描述的步骤表征前文库，然后进行文库构建、纯化和定量。荧光拟南芥标记系是通过将荧光蛋白与靶细胞类型中特异性表达的基因融合或使用增强子捕获系而开发的。根据荧光强度、前向散射和侧向散射单体成功分选荧光特异性标记原生质体。使用明场和荧光成像可视化分选的细胞。显示了约2， 000个木质部极周细胞，侧根原始细胞，内皮或皮层细胞以及侧根帽细胞的RNA测序文库的代表性结果。具有引物或接头二聚体峰的小尺寸文库在尺寸选择后仍可测序。大小分布异常的文库表示文库制备失败。分析了基因表达模式。将富集在四种根细胞类型中的任何一种中的基因聚集在一起，并在热图中描绘，显示不同细胞类型之间基因表达的特异性。通过正确的设门和分选以及防止RNA降解来高质量和纯度的靶细胞是成功构建文库的关键。对于单细胞模式下的RNA-seq，最近报道了几种与唯一分子标识符集成的方法，这些方法显着提高了性能。

Research

JoVE Journal

Biology

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

交配和四分体分离衣藻遗传分析

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

科研

生物学

Fluorescence Activated Cell Sorting of Plant Protoplasts

植物原生质体的荧光激活细胞分选

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

综合植物细胞壁成分分析（木质纤维素生物质）第一部分：木质素

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

一种新型的全基因组分析的贝叶斯变点算法的多元ChIPseq数据类型

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

植物表皮脂质聚酯单体的分离及成分分析

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...