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
Summary
帕金森病（PD），黑质（SNC）多巴胺能神经元退化，导致运动功能障碍。此处我们报告从表达eGFP的小鼠通过酪氨酸羟化酶（TH）的启动子序列驱动的培养腹侧中脑神经元，从培养收获个别荧光神经元，并使用RNA-SEQ测量它们转录的协议。
Abstract
在帕金森氏病（PD）有贯穿了在黑质致密多巴胺能神经元的显着退化脑中广泛的神经元丧失，导致运动徐缓，强直和震颤。在初级腹侧中脑（VM）的培养活多巴胺能神经元的使用荧光标记的识别提供了一种替代的方法来研究这些神经元的选择性脆弱而不依靠固定的细胞的免疫染色。在这里，我们分离，解离，并培养小鼠VM神经3周。然后，我们确定在使用的eGFP的荧光（由酪氨酸羟化酶（TH）启动子驱动）的培养物的多巴胺能神经元。单个神经元被收集到用玻璃微离心管。接下来，我们裂解收获的细胞，并进行cDNA合成和转座子介导的“tagmentation”，以产生单细胞RNA测序文库1，2，屁股=“外部参照”> 3，4，5。传递一个质量控制检查后，单细胞库进行测序和随后的分析进行测量的基因表达。我们报告个人多巴胺和中脑培养物中分离GABA能神经元转录组的结果。我们报告，收获和测序，该活的TH-eGFP的细胞的100％的多巴胺能神经元。这些技术将在神经科学和分子生物学广泛的应用。
Introduction
帕金森氏病（PD）是一种不可治愈的，年龄相关的神经变性疾病。此相对常见病症的起因仍然知之甚少。有广泛的神经元损失整个大脑，用在黑质（SNC）多巴胺能神经元的显着的神经元变性，导致运动徐缓，强直，震颤的诊断的临床特征。
含黑质多巴胺能神经元主要混合培养是帕金森氏病尤为重要。腹侧被盖区（VTA）多巴胺能神经元有牵连的奖励和上瘾。腹侧（VM）的主要混合胚胎培养同时包含黑质致密部和腹侧被盖区多巴胺（DA）神经元和GABA能神经元。 VM初级培养物可以是用于神经保护测定法有用并阐明多巴胺能神经元的选择性脆弱性。有识别基于形态学的培养多巴胺能细胞没有可靠的方法。他再我们开发技术来识别和收获单多巴胺能神经元，并构建单细胞，高产出的RNA测序文库。
我们报告单多巴胺和中脑培养物中分离GABA能神经元代表RNA转录组数据。该协议可以用于神经保护，神经变性，和药理学试验来研究在DA / GABA转录各种处理的影响。由于多巴胺能神经元代表主虚拟机的文化表达的神经元的一小部分，可靠地识别活的文化这些神经元的能力将实现单细胞研究的增强范围。这些新颖的技术将有助于理解发生在细胞水平上的机制的进展，并且可以具有应用在神经科学和分子生物学领域的其它地方。
Protocol
Representative Results
Discussion
在这里，我们分离使用荧光标记，然后研究以单细胞的RNA-SEQ每个小区由一个异质群体单细胞。我们报告，我们收获并测序活的TH-eGFP的细胞100％的确多巴胺能神经元，是根据以下三个DA相关基因的转录，TH，DDC和SLC6A3的存在。所有TH-EGFP阳性细胞通过RNA-SEQ的评估表达这三个基因。这是一致的与电生理研究显示，每TH-EGFP细胞也呈现出与多巴胺能神经元8,9相?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the U.S. National Institutes of Health (DA017279, AG033954, DA037743), the Michael J. Fox Foundation, and the Caltech Innovation Initiative funding the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. We thank Brian Williams for optimising the RNA-Seq library protocol and for providing assistance. We thank Henry Amrhein for computational training. We thank Barbara J. Wold for the use of her equipment and laboratory space. We thank Igor Antoshechkin for library sequencing, and for facility management.
Materials
| Papain | Worthington Biochemical Corporation | LS003126 | |
| Hanks’ Balanced Salt Solution (HBSS), 1X | Gibco | 14175-095 | |
| Donor Equine Serum | Thermo Scientific | SH30074.03 | |
| Bovine Serum Albumin (BSA) | Sigma | A7030-50G | |
| Medium: Neurobasal medium | Gibco | 21103-049 | |
| Culture media that contains a stabilized form of L-glutamine, L-alanyl-L-glutamine: GlutaMAX | Gibco | 35050-061 | 0.5 mM final concentration. |
| L-Ascorbic acid | Sigma | A7506 | |
| B27 | Gibco | 17504-044 | 50 X stock solution, 1 X final concentration. |
| 35 mm glass bottom dishes | MatTek | P35GC-1.5-10-C | |
| Poly-L-Ornithine | Sigma | P4957 | |
| Laminin | Sigma | L2020 | Stored at -20°C in 20 µL aliquots |
| Deoxyribonuclease I (DNase) | Sigma | DN25 | |
| Blue pipette tips | Sorenson Bioscience, | Inc. 10130 | |
| P1000 | |||
| 10 mm shallow Petri dish | VWR | 25384-324 | |
| 37°C Water bath | |||
| 37°C, 5% humidity incubator | |||
| 16% paraformaldehyde (PFA) | Electron Microscopy Sciences | 15710 | |
| Triton X-100 | Sigma | X100-500ML | |
| Donkey serum | Equitech | SD-0100HI | 100% stock solution, 1% final concentration. |
| Standard Pattern surgical scissors | Fine Science Tools | 14000-14 | |
| Forceps – 2×3 teeth | Fine Science Tools | 11022-14 | |
| Forceps – Dumont #55 | Fine Science Tools | 11295-51 | |
| Forceps – Dumont #5 | Fine Science Tools | 11252-23 | |
| 10X PBS, pH 7.4 | Gibco | 70011-044 | |
| Dulbecco's Phosphate Buffered solution (DPBS) 1X | Gibco | 14190-144 | 500 ml |
| 21 guage needle | BD PrecisionGlide Needle | 305167 | 100 needles |
| Micropipette puller | Sutter Instrument. | model P-87 | |
| Micromanipulator | Sutter Instrument | MP-285 | |
| Sequencing Kit: SMARTer Ultra Low RNA Kit for Illumina Sequencing | Clontech | 634936 | 100rnxs,incl Advantage2PCR Kit |
| oligonucleotide (12 µM): SMARTer IIA oligonucleotide (12 µM) | From the SMARTer Kit | ||
| Reverse transcriptase (100 units) | SMARTcribe reverse transcriptase | From the SMARTer Kit | |
| Primer 1 | 3’ CDSIIa primer | From the SMARTer Kit | |
| Primer 2 | IS PCR primer | From the SMARTer Kit | |
| 50X 2 polymerase mix | 50X Advantage 2 polymerase mix | From the SMARTer Kit | |
| 10X PCR buffer | 10X Advantage 2 PCR buffer | From the SMARTer Kit | |
| RNA Spikes | ThermoFisher | 4456740 | |
| PCR cooler rack | IsoFreeze PCR cooler rack. | ||
| DNA Sample Preparation Kit | (Illumina/Nextera) Nexter | FC-121-1030 | 24 Samples |
| PCR master mix | Nextera PCR master mix (NPM) | From the Nextera Kit | |
| PCR primer cocktail (PPC) | Nextera PCR primer cocktail (PPC) | From the Nextera Kit | |
| Fluorometer | The Qubit ThermoFisher | Q33216 | |
| HS DNA BioAnalyzer kit | Agilent | 5067-4626 | 110rxns |
| Electrophoresis system | Agilent 2100 Bioanalyzer. | G2938C | |
| Magnetic beads: Agencourt AMPure | Beckman Coulter | A63880 | 5ml |
| RNAse wipe: RNAse Zap wipes | Ambion | AM9786 | Size 100 Sheets |
| Qubit ds HS Assay Kit | Molecular probes | Q32854 | 500 assays |
| Gel extraction kit: Qiaquick Gel Extraction Kit | Qiagen | 28704 | |
| Glass capillary tubing | (Kimax-51, 1.5–1.8 mm o.d.) | ||
| Microforge | Narishige (or equivalent) | ||
| Sequencer | Illumina HiSeq instrument | ||
| GENSAT tyrosine hydroxylase-eGFP mouse strain | MMRRC stock number: 000292-UNC | Stock Tg(Th-EGFP)DJ76Gsat/Mutant Mouse Regional Research Center |
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