JoVE Journal > Developmental Biology
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Abstract
Introduction
Protocol
Results
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Acknowledgements
Materials
References
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Developmental Biology

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

Published: May 14, 2020
doi:
10.3791/60919
, ,
1Department of Plant and Microbial Biology,University of Zurich, 2Biophore – UNIL, 3Laboratory of Cell and Molecular Biology, Institute of Biology,University of Neuchâtel

Summary

翻译核糖体亲和纯化（TRAP）提供了以最少的器官和组织处理来解剖发育方案的可能性。该协议从绿色荧光蛋白（GFP）标记的核糖体亚组细胞中产生高质量的RNA。下游分析工具（如 qRT-PCR 或 RNA-seq）可显示组织和细胞类型特定的表达特征。

Abstract

在本文中，我们给出了通过翻译核糖体亲和纯化（TRAP）方法和连续优化的低输入库制备从不同阿拉伯生物基系thaliana根细胞类型获取的翻译数据的实践说明。

作为起始材料，我们使用植物生产线，使用适当的促进剂，以细胞类型特定的方式表达GFP标记核糖蛋白RPL18。在免疫纯化和RNA提取之前，组织被卡扣冷冻，从而保持组织的完整性，同时允许以高时间分辨率执行时间序列研究。值得注意的是，细胞壁结构保持不变，这是替代程序的一个主要缺点，例如荧光激活细胞排序方法，依靠组织原面分离不同的细胞群。此外，无需像激光捕获微解剖技术那样组织固定，从而获得高质量的RNA。

然而，从细胞亚群的取样，仅分离多体相关RNA，严重限制了RNA的产生。因此，有必要应用足够灵敏的库制备方法，以便RNA-seq成功采集数据。

TRAP 为植物研究提供了理想的工具，因为许多开发过程都涉及与细胞壁相关和机械信号通路。利用促进剂瞄准特定细胞群，正在缩小器官和单细胞水平之间的差距，而单细胞水平又因分辨率小或成本非常高而受到影响。在这里，我们应用TRAP来研究侧根形成的细胞-细胞通信。

Introduction

在下一代测序技术日益应用的推动下，空间分辨率在发育生物学中可以增强。当代研究旨在解剖组织到专门的细胞类型，如果不是单细胞水平11，2，3，4。2,3,4为此，在过去五十年中，已设计了大量不同的方法（见图1A）5、6、7、8、9、10、11、12、13、14、15。,14,,7,8,9,10,11,12,135,6 Figure 1A15

植物科学中的许多工具都是对动物研究首创的技术的适应。我们在此处详细介绍的方法不是这样。2005年，凭借蛋白质翻译的强势背景，贝利-塞雷斯实验室开始设计核糖蛋白，用于后续的亲和力纯化16。因此，它们可以避免耗时和劳动密集型的多体分析，这是基于超中心与蔗糖梯度，并用于评估翻译核糖体自20世纪60年代17，18。17,该方法自即被称为转化核糖体亲和纯化（TRAP）16。在成功的植物翻译研究后，Heiman19等人将其应用扩展到酵母20、果蝇21、异种鱼23、24。,2422

虽然模型系统的遗传改造是TRAP的先决条件，它将其应用限制在适应遗传转化的物种，人们可以同时利用这种异议，以针对特别感兴趣的细胞子集，否则极难从完整的组织/器官25（例如，在小鼠大脑中高度分支的树突状细胞或受感染的植物组织中的真菌催眠）。在植物中，所有细胞都通过细胞壁固定到位，形成静水骨架26的基础。为了将植物细胞从这个基质中释放出来，科学家要么通过激光捕获微切（LCM）27从周围组织中物理地切27出细胞，要么对细胞壁进行酶消化28。在后一个细胞中，所谓的正代，感兴趣的种群被荧光标记，可以通过荧光激活细胞分拣（FACS）7分离。7LCM通常需要将样品固定并嵌入蜡中，这最终会降低其RNA29的质量。基于FACS的方法产生高质量的RNA，但提供过程本身引入了基因表达的差异30和具有修饰和厚的继生细胞壁组织是出了名的难以治疗。此外，植物中的许多发育过程被认为是依靠机械传输的信号，因此细胞壁的完整性是最重要的31。两种方法，使用快捷方式，通过在核水平上操作来规避细胞分离，是荧光激活核分拣（FANS）和特定细胞类型（INTACT）标记的核分离。与TRAP一样，它们使用细胞类型特定的促进器来标记核，然后通过排序或拉下获得丰富，分别为,8，15。所有这些方法的一个主要挑战是从组织中的细胞子集获得足够的RNA材料。由于 TRAP 只捕获蜂窝 RNA 的一小部分，因此样本采集是一个相当大的瓶颈。因此，需要特别敏感的库准备协议来生成低输入量的高质量数据。

自建立以来，TRAP 要么与 DNA 微阵列结合使用，要么随着近年来测序成本的显著下降，RNA-seq 10、32、33 。10,32,33在Sablok等人34日回顾了许多研究问题。我们相信，在未来几年中，将会有更多的报告，因为结合不同的启动子来靶向特定的细胞类型时，该技术非常通用。最终，即使以可诱导的方式完成，也可以结合探测植物对许多生物和非生物应激因素的反应。此外，在没有稳定的转基因线的情况下，毛茸茸根表达系统也已成功用于在番茄和药质35，36,36中执行TRAP。

Figure 1
图1：翻译核糖体亲和力纯化（TRAP）补充了”经济学”分析组合。A.通过大量方法或组合，可以提高分析精度，甚至单细胞甚至亚细胞分辨率。该计划概述了目前在植物和动物领域可用的工具。细胞分辨率下的组织收集可以通过LCM或FACS等协议实现，然后与标准转录组或多体分析/翻译分析耦合。TRAP 和 INTACT 将组织捕获和 RNA 分离结合在一起，因为它们基于表上标记。然而，INTACT只样本细胞核，因此，构成转录分析的特殊情况。一个小兔子图标标记了动物领域新开发的方法：虽然SLAM-ITseq和Flura-seq依赖于在表达放任性酶的细胞中具有改性尿素碱的新生RNA的代谢靶向，Slide-seq使用带有DNA条形码的涂层玻璃幻灯片，在细胞范围内提供位置信息。APEX-seq 中遵循接近标记方法，在特定亚细胞隔间中对 RNA 进行采样。值得注意的是，分辨率提高通常需要生成转基因材料（星号），因此这些方法主要用于模型物种。TRAP 特别适用于涉及细胞壁 （CW） 或机械信号的植物科学研究，以及难以从其 CW 基质中释放的细胞物种。B. TRAP程序的详细湿实验室步骤：在不同细胞类型（如根内皮）中表达GFP标记核糖蛋白的幼苗在培养皿上生长7天，通过卡扣冷冻收获根料。在通过离心对碎片进行颗粒化之前，从均质原油提取物中采集总RNA控制样本。磁性抗GFP珠被添加到清除的提取物中，以执行免疫沉淀。孵化和三洗步骤后，通过酚-氯仿提取直接获得多体相关RNA（TRAP/多体RNA）。LCM：激光捕获微切解剖，FACS/FANS：荧光激活细胞/核分拣， APEX-seq：基于工程抗坏血剂过氧化物酶的方法，INTACT：特定细胞类型中标记的核分离，SLAM-ITseq：用于组织RNA代谢测序的THiol（SH）相关烷基化，Flura-seq：氟酸酯标记RNA测序（用Biorender.com创建）请点击这里查看这个数字的较大版本。

本文的目的是提供 TRAP 方法的详细说明，以突出显示关键步骤，并为可能的库准备方法提供指导。

通用 TRAP 实验将基本包括以下步骤（另见图1B）：（1）植物材料的准备，包括核糖体标记结构的克隆、转基因生产线的生产和选择、种子的生长和扩充、灭菌和电镀以及应力应用/处理（可选）和组织收获;（二）免疫纯化，包括组织均质化、清纯粗提取物、珠洗、免疫净化、洗涤步骤;（3） RNA提取和质量评估;（4） 图书馆准备。

阿拉伯植物根自引进示范植物37、38,38以来，一直是研究植物发展的示范系统。在这里，TRAP的应用在植物横向根开发的背景下被展示出来。在植物中，整个根系的建立依赖于这个程序的执行，因此对生物体39的生存非常重要。在Arabidopsis中，侧根起源于位于赛莱默血管旁边的围轮组织，因此被称为木质杆围轮（XPP;参见图2C）40。40一些XPP细胞，位于根的深处，获得一个创始人细胞的身份，并在局部荷尔蒙触发下，开始通过肿胀和分裂41开始增殖。然而，由于存在刚性细胞壁基质，这一过程对周围的组织施加机械应力。特别是，覆盖内皮受影响，因为它是在横向根生长轴42，43，4443,44的方式。42事实上，新形成的原体将不得不通过覆盖的内皮细胞（图2C2）生长，而皮层和表皮细胞只是被推到一边，使原体最终出现45，46。,46我们实验室最近的工作表明，内皮病正在积极推动适应围周期的增殖。靶点阻塞内皮激素信号足以抑制甚至第一分裂在XPP细胞47。因此，围周期-内皮斯通信是阿拉伯细胞横向根系发展的一个早期检查点。然而，不知道这个相声是如何进行的。为了解开这个谜团，我们选择了TRAP-seq方法来瞄准XPP和内皮细胞。为了丰富横向根程序中的细胞，我们通过外生应用辅助辛酸（1-naph甲酸，NAA）48来模拟荷尔蒙触发，同时允许暂时解决侧根形成48的初始阶段。

Protocol

1. 转基因克隆、转基因生产线生产和选择 在适当的入口矢量中克隆所选择的启动子。使用基于重组的克隆方法（材料表），并使用pDONRP1-P249中基于重组的克隆重新组合pDONRP4-P1r.克隆RPL18（具有亲和力标记或荧光蛋白）。 使用 FAST-RED 选择盒50将包含RPL18的入口矢量与包含启动子的入口矢量在两个片段重组反应中合并到适当的目标…

Representative Results

在质量评估方面，应探讨上述过程的几个中间步骤：植物中的表达模式验证、分离的多体层RNA的质量控制以及最终库。此外，还可以使用已知标记基因进行qRT-PCR，以确认对治疗条件的反应或微调实验条件。 GFP信号分布的共聚焦分析为了检查内皮和XPP表达模式，我们通过共聚焦显微镜分析了pELTP：：：GFP-RPL18<…

Discussion

验证 RPL18 本地化模式
避免任何 TRAP 实验的数据被误解的关键，是标记的核糖体子单位的正确表达模式。因此，将 GFP 作为表位标记与 RPL18 的上部标签非常优雅地允许验证所需的表达模式，并连续从同一组织中下拉多体分数。更多侵入性的方法，以确保适当的促进者模式遵循焦和梅耶罗维茨2010年，这需要GUS染色和在Tian等人2019年依靠免疫染色与抗FLAG抗体58，59。<sup…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们要感谢苏黎世遗传多样性中心的让-克洛德·沃尔瑟在该项目的早期阶段提供重要的专家建议。Vermeer 实验室的工作得到了瑞士国家科学基金会 （SNSF） 授予 JEMV 的 SNF 教授授予 （PP00P3_157524） 和 R’EQUIP 设备赠款 （316030_164086） 的支持。

Materials

Sterilization
bleach, 13% Sigma 71696
beaker VWR 214-1172/74/75
desiccator with porcelaine plate (DURAN) Sigma/Merck Z317454-1EA/Z317594-1EA
EtOH, p.a. Honeywell 02860-1L
HCl, 37% Roth 4625.1
Tween 20 Sigma P9416
Plate growth + harvesting
MS salts, basal salt mixture, incl. MES buffer Duchefa M0254
agar plant for cell culture Applichem/Panreac A2111.1000
DMSO Sigma D4540
forcepts Rubis Switzerland 5-SA model
KOH Fluka 60370
micropore/surgical tape 3M 1530-0
NAA Duchefa N0903
petri dishes 120×120 mm Greiner bio-one 688102
scalpel VWR/Swann-Morton 233-5454
tissues, neutral, two-layered any supplier of your choice
Immunoprecipitation
GFP-beads: gtma-100 GFP-Trap_MA Chromotek e.g. gtma-100
Brij-35 Sigma P1254-500G
centrifuge tubes (in accordance with centrifuge) Beckman Coulter 357001
Chloramphenicol Applichem C0378-25G
cotton gloves VWR 113-7355
Cycloheximide, HPLC grade Sigma 01810-1G
DEPC VWR E174 might have long delivery times
DTT Fluka 43815
EGTA Sigma 3054.3
homogenizers DUALL 23 KONTES GLASS CO (via VWR) SCERSP885450-0023 (set) SCERSP885451-0023 pestle only – SCERSP885452-0023 cylinder only; long delivery times
Igepal CA-360 Sigma I3021-100ml
KCl Sigma 60130
MgCl2 hexahydrat Roth 2189.2
mortar and pestle VWR 470148-960 & 470019-978
PMSF Roche 10 837 091 001
Polyoxyethylene-(10)-tridecylether/PTE Sigma P2393-500G
RNase-free water Roth T143.3
RNAZap Thermo Fisher AM9780/AM9782 for cleaning surfaces
Tris, >99.3% Roth AE15.3
Triton X-100 Fluka T8787-250ml
Tween 20 Sigma P9416-100ml
RNA extraction
2-Propanol, p.a. Sigma 33539-1L-GL-R
Chloroform, HPLC grade Scharlau CL02181000
EtOH, p.a. Honeywell 02860-1L
low-retention microcentrifuge tubes, 1.5 ml Eppendorf/Sigma Z666548-250EA LoBind
RNase-free DNase set Qiagen 79254
RNeasy MiniElute Cleanup Kit Qiagen 74204
TRIzol reagent ThermoFisher/Ambion 15596018
Library preparation
15/50 mL Tube Magnetic Separator Abraxis PN 472250
AMPure beads Beckman Coulter A63881
Index Kit A Illumina FC-131-2001
Index Kit D Illumina FC-131-2004
neodymium magnets Amazon/other 6 x 1.5 mm range: N42 (NdFeB)
Nextera XT kit Illumina FC-131-1024/1096 https://emea.support.illumina.com/
PCR strips ThermoScientific AB-0266
SMARTer v4 kit Takara Bioscience 634892 https://www.takarabio.com/
Bioanalyzer Agilent 2100 Bioanalyzer Instrument specialized equipment for RNA/DNA quality control
Tapestation Agilent 4200 Tapestation Instrument specialized equipment for RNA/DNA quality control
Fragment Analyzer Agilent 5400 Fragment Analyzer System specialized equipment for RNA/DNA quality control (high throughput)
LabChip PerkinElmer LabChip GX Touch Nucleic Acid Analyzer specialized equipment for RNA/DNA quality control (high throughput)
Qubit 4 Fluorometer ThermoFisher Q33239 specialized equipment for RNA/DNA concentration determination
qRT-PCR
GATA23 Microsynth fwd: AGTGAGAATGAA
AGAAGAGAAGGG;
rev: GTGGCTGCGAAT
AATATGAATACC
GH3.3 Microsynth fwd: CAAACCAATCCT
CCAAATGAC;
rev: ACTTATCCGCAA
CCCGACT
LBD29 Microsynth fwd: TCTCCAACAACA
GGTTGTGAAT;
rev: AAGGAGCCTTAG
TAGTGTCTCCA
UBC21 Microsynth fwd: TGCGACTCAGGG
AATCTTCT;
rev: TCATCCTTTCTT
AGGCATAGCG
SsoAdvanced Universal SYBR Green Bio-Rad #172-5270
iScript Adv cDNA Kit Bio-Rad #172-5038
miscellaneous
Falcon tubes 15 ml, Cellstar Greiner bio-one 188261
Falcon tubes 50 ml, Cellstar Greiner bio-one 210261
filter tips 1 ml Axygen TF-1000-R-S
filter tips 10 µl Axygen TF-10-R-S
filter tips 100 µl Axygen TF-100-R-S
filter tips 20 µl Axygen TF-20-R-S
filter tips 200 µl Axygen TF-200-R-S
microcentrifuge tubes 1.5 ml SARSTEDT 72.690.001
Propidium iodide Sigma P4170-100MG
sequencing company Novogene en.novogene.com
DOWNLOAD MATERIALS LIST

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Ribosome Affinity Purification (TRAP)Arabidopsis ThalianaRoot DevelopmentCell Type-specificPolysome RNALow-input RNALateral Root FormationCell-cell CommunicationCell WallMechanical SignalingSeed SterilizationExogenous TreatmentNAA

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Thellmann, M., Andersen, T. G., Vermeer, J. E. Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale. J. Vis. Exp. (159), e60919, doi:10.3791/60919 (2020).
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