植物细胞类型的分离和转录组分析
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 0.22 µm strainer | Sorfa | 622110 | |
| Agar | Yeasen | 70101ES76 | |
| Agilent fragment analyzer | Aglient | Aglient 5200 | |
| Agilent high-sensitivity DNA kit | Aglient | DNF-474-0500 | |
| Ampure XP beads | BECKMAN | A63881 | |
| Betaine | yuanye | S18046-100g | |
| Bleach | Mr Muscle | FnBn83BK | 20% (v/v) bleach |
| BSA | sigma | 9048-46-8 | |
| CaCl2 | yuanye | S24109-500g | |
| Cellulase R10 | Yakult (Japan) | 9012-54-8 | |
| Cellulase RS | Yakult (Japan) | 9012-54-8 | |
| Centrifuge tube (1.5 mL) | Eppendolf | 30121589 | |
| DNase, RNase, DNA and RNA Away Surface Decontaminants | Beyotime | R0127 | |
| dNTPs (10 mM) | NEB | N0447S | |
| DTT (0.1 M) | invitrogen |
18090050 | |
| Ethanol | Sinopharm Chemical Reagent Co., Ltd | 100092680 | |
| FACS | BD FACS Melody | BD-65745 | |
| FACS | Sony | SH800S | |
| Filter tip (1000 µL) | Thermo Scientific | TF112-1000-Q | |
| Filter tip (200 µL) | Thermo Scientific | TF140-200-Q | |
| Filter tip (10 µL) | Thermo Scientific | TF104-10-Q | |
| Filter tip (100 µL) | Thermo Scientific | TF113-100-Q | |
| Fluorescent microscope | Nikon | Eclipse Ni-E | |
| Four-Dimensional Rotating Mixer | Kylin -Bell | BE-1100 | |
| Hemicellulase | sigma | 9025-56-3 | |
| IS PCR primer | 5'-AAGCAGTGGTATCAACGCAGAG T-3' |
||
| KAPA HiFi HotStart ReadyMix(2X) | Roche | 7958935001 | |
| KCl | Sinopharm Chemical Reagent Co., Ltd | 7447-40-7 | |
| Macerozyme R10 | Yakult (Japan) | 9032-75-1 | |
| Magnetic separation stand | invitrogen | 12321D | |
| Mannitol | aladdin | 69-65-8 | |
| MES | aladdin | 145224948 | |
| MgCl2 | yuanye | R21455-500ml | |
| Microcentrifuges | Eppendorf | Centrifuge 5425 | |
| Micro-mini-centrifuge | Titan | Timi-10k | |
| MS | Phytotech | M519 | |
| Nextera XT DNA Library Preparation Kit | illumina | FC-131-1024 | |
| oligo-dT30VN primer | 5'-AAGCAGTGGTATCAACGCAGAG TACTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTVN-3' |
||
| PCR instrument | Thermal cycler | A24811 | |
| Pectolyase | Yakult (Japan) | 9033-35-6 | |
| Plant marker lines | Nottingham Arabidopsis Stock Centre (NASC) | ||
| Qubit 1x dsDNA HS Assay Kit | invitrogen | Q33231 | |
| Qubit 2.0 fluorometer | invitrogen | Q32866 | |
| RNase inhibitor | Thermo Scientific | EO0382 | |
| RNase-free water | invitrogen | 10977023 | |
| Solution A | 400 mM mannitol, 0.05 % BSA , 20 mM MES (pH5.7), 10 mM CaCl2, 20 mM KCl | ||
| Solution B | 1 % (w/v)cellulase R10, 1 % (w/v) cellulase RS, 1 % (w/v)hemicellulase, 0.5 % (w/v)pectolyase and 1 % (w/v) Macerozyme R10 of in a fresh aliquot of solution A | ||
| Sterile pestle | BIOTREAT | 453463 | |
| Strainer (40 µm ) | Sorfa | 251100 | |
| Superscript enzyme (200 U/µL) | invitrogen | 18090050 | |
| SuperScript VI buffer (5x) | invitrogen | 18090050 | |
| T0est tube (5 mL) | BD Falcon | 352052 | |
| Thin-walled PCR tubes with caps (0.5 mL) | AXYGEN | PCR-05-C | |
| Triton X-100 | Sangon Biotech | A600198-0500 | |
| TSO primer | 5'-AAGCAGTGGTATCAACGCAGAG TACATrGrG+G-3' |
||
| Vortex | Titan | VM-T2 |
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Cite This Article
Zhang, J., Ahmad, M., Xie, R., Gao, H. Isolation and Transcriptome Analysis of Plant Cell Types. J. Vis. Exp. (194), e64913, doi:10.3791/64913 (2023).