激光显微切割，用于与物种无关的单组织应用
Summary
描述了一种使用激光显微切割来分离单个线虫组织以进行RNA测序的方案。该协议不需要物种特异性遗传工具包，允许在单组织样本水平上比较不同物种之间的基因表达谱。
Abstract
单细胞方法彻底改变了特定细胞类型转录组的分析。然而，它们通常需要物种特异性遗传“工具包”，例如驱动荧光蛋白组织特异性表达的启动子。此外，破坏组织以分离单个细胞的方案将细胞从其天然环境中移除（例如，来自邻居的信号传导），并可能导致应激反应或与天然基因表达状态的其他差异。在本方案中，激光显微切割（LMD）被优化以分离单个线虫尾尖，用于研究雄性尾尖形态发生期间的基因表达。
LMD允许在不需要细胞破坏或物种特异性工具包的情况下分离动物的一部分，因此适用于任何物种。随后，单细胞RNA-seq文库制备方案（如CEL-Seq2）可以应用于LMD分离的单组织，并使用标准管道进行分析，前提是该物种有一个注释良好的基因组或转录组。这些数据可用于确定转录组在不同物种中形成组织的基础的保守性或差异性。
局限性包括切除感兴趣组织和样本数量的能力。功率分析表明，80%的功率只需要每个条件70个尾尖。需要紧密同步发育才能在同一发育阶段获得这么多的动物。因此，还描述了一种以1小时间隔同步动物的方法。
Introduction
线虫 – 特别是与秀 丽隐杆线虫模型系统相关的横纹线虫 – 是进化发育生物学（EDB）的一组奇妙的动物，原因有很多1，2。其优点包括细胞数量少，细胞谱系明确且一致，透明度高，易于培养和饲养。还有许多可用的资源，包括多个物种的高质量基因组和 秀丽隐杆线虫，广泛的分子遗传工具和有关发育，遗传学，解剖学和生理学的知识3，4，5，6。
与许多其他生物体一样，表征单个组织或单个细胞中转录组动力学的能力彻底改变了秀丽隐杆线虫7，8，9，10的发育分析。能够比较线虫之间的单细胞转录组同样会使用这些生物体改变EDB。例如，这种比较将深入了解基因调控网络如何为保守的字符（性状），已经分化的字符或独立进化的字符进化。
然而，从线虫中分离出特定的组织或细胞是最大的挑战之一。对于许多生物体，单细胞可以从组织中解离并以无偏倚的方式收获，或者可以用荧光蛋白的组织特异性表达进行标记，并通过荧光活化细胞分选（FACS）11进行分类。在秀丽隐杆线虫中，细胞的高通量（HTP）分离主要局限于胚胎，因为坚韧的外部角质层（和流体静压骨架）阻碍了细胞从幼虫和成虫的分离。为了解决这一挑战，一些方法在整个秀丽隐杆线虫中采用了遗传工具，例如组织特异性mRNA标记12，以及影响细胞类型13的野生型和突变体之间的差异表达比较。最近的方法通过溶解角质层来分离细胞核14或整个细胞8，9，15来克服这一挑战。然而，细胞分离和细胞培养具有明显的缺点，即细胞从其自然发育或解剖学环境中移除 – 例如，远离细胞 – 细胞信号传导和与细胞外基质的接触 – 这预计会影响基因表达谱15。此外，遗传工具和组织特异性标记是物种特异性的（即，它们只能用于秀丽隐杆线虫）。
LMD提供了一种在不破坏细胞自然环境的情况下分离组织的替代方法。对于EDB来说，LMD还允许比较来自不同物种的同源组织的转录组，而无需物种特异性遗传工具包（如果这些物种的基因组或全转录组序列可用）。LMD涉及通过直接显微镜观察和使用集成到显微镜光学元件中的激光微束来靶向组织，以切割和收获（捕获）感兴趣的组织16。LMD的局限性在于它不利于非常HTP的方法（尽管如本实验方案中所述，尾尖的转录谱对于约70个样品是稳健的），某些样品可能难以剖析，并且切割仅限于激光的精度和可以在显微镜中可视化的内容。
本方案的目的是描述LMD（然后是单组织RNA-Seq）如何用于从线虫获得阶段和组织特异性转录组数据。具体而言，它证明了LMD用于从 秀丽隐杆线虫的第四阶段幼虫（L4）中分离尾尖。然而，这种方法可以适应其他组织，当然还有不同的物种。
在 秀丽隐杆线虫中，有4个细胞在雄性和雌雄同体中形成尾尖。在雄性的L4阶段 – 但不是雌雄同体 – 尾尖细胞改变其形状并向前和向内迁移。这个过程也发生在一些但不是所有其他横纹线虫物种中。因此，尾尖是有性二态形态发生进化的良好模型。由于其位置，尾尖也很容易被LMD隔离。
为了从尾尖获得转录组图谱，本方案使用CEL-Seq2，这是一种为单细胞17，18开发的RNA-seq方法。该方法对于LMD衍生的组织有几个优点。CEL-Seq2 具有高灵敏度和高效率，使用独特的分子标识符 （UMI） 对 mRNA 读数进行直接定量， 通过体外 转录确保线性扩增，以及条形码（允许对单个组织样品进行多路复用）。CEL-Seq2的唯一限制是恢复的读数偏向于mRNA的3’末端，因此无法区分大多数亚型。
Protocol
Representative Results
Discussion
该方法的关键步骤
如果操作正确，此处描述的方法将使用相对较少的激光解剖样品（本例中有70个尾尖）获得稳健的RNA图谱。然而，对于来自发育中的动物的样品，紧密同步对于减少样品之间的变异性至关重要。因此，该协议建议使用删除出雏方法进行蠕虫病毒同步。在这里，研究人员可以确定并精确控制个体之间的年龄差异（本方案中的1小时）。此外，孵化方法适用于任何物种?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作由NIH（R01GM141395）和NSF（1656736）资助DF和NIH奖学金（F32GM136170）到AW。 图 1 是在 BioRender.com 的帮助下创建的。
Materials
| 0.5 µM PEN membrane glass slides RNase free | Leica | 11600288 | for LMD |
| 500 µL PCR tubes (nuclease-free) | Axygen | 732-0675 | to cut the tail tips into |
| Compound microscope with 40x objective and DIC | any | to check age of worms | |
| Desktop humidifier | any | ||
| Dissection microscope with transmitted light base | any | for all worm work | |
| glass pasteur pipets | any | handle of worm pick | |
| glass slides and coverslips | any | to check age of worms | |
| LMD6 microdissection system | Leica | multiple | to cut tail tips |
| LoBind tubes 0.5 mL | Eppendorf | 22431005 | |
| M9 Buffer | Recipe in WormBook | ||
| Methanol 99.8% | Sigma | 322415 | to fix worms |
| NGM growth medium | US Biological | N1000 | Buffers and salts need to be added: Recipe in WormBook |
| P10 pipette variablle volume | e.g. Gilson | ||
| P1000 pipette variable volume | e.g. Gilson | ||
| P2 pipette variable volume | e.g. Gilson | ||
| Pipette tips 1,000 µL | any | ||
| Pipette tips 1-10 µL filtered | any | ||
| platinum iridium wire | Tritech | PT-9010 | to make worm pick |
| sterile and nuclease-free 1 mL centrfuge tubes | any | ||
| Tween 20 | Sigma | P9416 | Add a very small amount to M9 buffer to prevent worms from sticking to the pipet tips |
| vented 6 mm plastic Petri dishes | any | ||
| For CEL-Seq2 | |||
| 4200 TapeStation System with reagents for high-sensitivity RNA and DNA detection | Aligent | automated electrophoresis system | |
| AMPure XP beads | Beckman Coulter | A63880 | DNA cleanup beads |
| Bead binding buffer 20% PEG8000, 2.5 M NaCl | |||
| CEL-Seq2 primers (see Table S1) | Sigma Genosys Mastercycler Nexus GX2 Eppendorf | 6335000020 | Thermal cycler with programmable lid and block for 200 µl tubes. |
| DNA Polymerase I (E. coli) | Invitrogen | 18052-025 | |
| dNTP mix 10 mM | any | ||
| E. coli DNA ligase | Invitrogen | 18052-019 | |
| Ethanol | |||
| ExoSAP-IT For PCR Product Clean-Up | Affymetrix | 78200 | exonuclease solution |
| MEGAscript T7 Transcription Kit | Ambion | AM1334 | For step 4.6.1 |
| Nuclease-free water | any | ||
| Phusion High-Fidelity PCR Master Mix with HF Buffer | NEB | M0531 | PCR mix step 4.9.7 |
| random hexamer RT primer GCCTTGGCACCCGAGAATTCCA NNNNNN |
IDT | a primer with 6 nucleotides that are random | |
| RNA Fragmentation buffer | NEB | E6150S | |
| RNA Fragmentation stop buffer | NEB | E6150S | |
| RNA PCR Index Primers (RPI1–RPI48) | Illumina, NEB, or IDT | RPIX in protocol step 4.9.7, sequences available from Illumina | |
| RNAClean XP beads | Beckman Coulter | A63987 | |
| RNase AWAY Surface Decontaminant | Thermo Scientific | 7000TS1 | or any other similar product |
| RNaseH (E. coli) | Invitrogen | 18021-071 | |
| RNaseOUT Recombinant Ribonuclease Inhibitor | Invitrogen | 10777-019 | |
| Second strand buffer | Invitrogen | 10812-014 | |
| Superscripit II | Invitrogen | 18064-014 | reverse transcriptase |
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