脑类器官中单细胞和单核转录组的生成和下游分析
概要
在这里，我们介绍了一种使用单细胞和单核RNA测序生成和下游分析人脑类器官的综合方案。
Abstract
在过去的十年中，单细胞转录组学已经取得了显着发展，并成为同时分析单个细胞基因表达谱的标准实验室方法，从而可以捕获细胞多样性。为了克服难以分离的细胞类型带来的限制，可以使用一种旨在恢复单核而不是完整细胞的替代方法进行测序，使单个细胞的转录组分析普遍适用。这些技术已成为大脑类器官研究的基石，使它们成为发育中的人类大脑的模型。利用单细胞和单核转录组学在脑类器官研究中的潜力，该协议提供了一个分步指南，涵盖了类器官解离、单细胞或细胞核分离、文库制备和测序等关键程序。通过实施这些替代方法，研究人员可以获得高质量的数据集，从而能够识别神经元和非神经元细胞类型、基因表达谱和细胞谱系轨迹。这有助于对塑造大脑发育的细胞过程和分子机制进行全面研究。
Introduction
在过去的几年中，类器官技术已成为培养器官样组织的一种有前途的工具1,2,3。特别是对于不容易接触到的器官，如人脑，类器官提供了深入了解发育和疾病表现的机会4.因此，大脑类器官已被广泛用作实验模型，用于研究各种人类脑部疾病，包括发育、精神疾病甚至神经退行性疾病 4,5,6。
随着单细胞转录组分析技术的出现，原代人体组织和复杂的体外模型可以以前所未有的粒度水平进行研究，为健康和疾病中细胞亚群水平上的基因表达变化提供机制见解，并为新的假定治疗靶点提供信息7,8,9.通过利用单细胞转录组分析来评估细胞组成、可重复性和大脑类器官技术的保真度，类器官领域取得了进展 10,11,12。单细胞 RNA 测序 （scRNA-seq） 使细胞分类和患病类器官中的遗传失调鉴定成为可能13,14。重要的是，正是类器官组织的复杂性使得有必要实施能够分析单个细胞的技术。使用批量转录组分析（批量 RNA 测序）等方法对类器官进行表征会导致细胞异质性和基因表达谱被掩盖，这些异质性和基因表达谱在复杂组织内的所有类型细胞中均被平均，最终限制了我们对健康和疾病中类器官发育过程中正在进行的过程的理解 15,16,17.随着scRNA-seq方法的不断发展，越来越多的图谱正在被创建，例如Uzquiano等人的Allen Brain Atlas或Single cell atlas of human brain organoids等资源18。
从脑类器官中成功实现 scRNA-seq 依赖于有效分离和捕获完整细胞。由于脑类器官的解离以获得单个细胞是基于酶消化的，因此它可以通过诱导应激和细胞损伤来影响基因表达模式19,20。因此，将组织解离成单个细胞是最关键的一步。另一种方法是单核 RNA 测序 （snRNA-seq），它有助于从新鲜和冷冻组织中无酶提取细胞核21,22。然而，从组织中分离细胞核会带来其他挑战，例如目标细胞类型的富集以及与细胞相比细胞核的 RNA 含量低。
脑类器官的转录组研究通常使用 scRNA-seq 10,18,23 进行。然而，单核的分离可能提供了一种正交和补充方法来研究类器官的转录组学特征。在这里，我们介绍了一个用于脑类器官scRNA和snRNA-seq的工具箱，并讨论了获得最佳质量测序数据的关键点。
Protocol
所描述的方案在Max Delbrück分子医学中心（批准号：138/08）的生物安全1级实验室中进行，符合要求并符合欧盟和国家的研究伦理规则。 1. 从诱导多能干细胞 （iPSCs） 衍生出前脑类器官 注：该协议已针对在来自不同公司的各种干细胞培养基中培养的几种不同iPSC系进行了测试（表1）。前脑类器官的产生高度依赖于高质量的 iPSC 和 60%-…
Representative Results
为了使用 scRNA-seq 和 snRNA-seq 研究脑类器官的细胞类型组成，在培养 30 天后收获脑类器官，因为该阶段的类器官已经表现出由中间祖细胞包围的祖细胞和早期神经元组成的神经上皮环 4,18。在整个生长和培养过程中监测类器官的质量对于获得可靠的单细胞和单核数据至关重要。 类器官是通过将iPSC聚集到胚状体中而形成的（<strong class…
Discussion
单细胞和单细胞核的转录组学分析已成为理解复杂组织内基因调控机制的关键工具。这两种方法都可以对大脑类器官进行转录组研究。为了确保实验的整体成功，起始材料的质量具有高度相关性。因此，有必要定期切割类器官以防止形成坏死核心26。也可以通过气液界面培养物来消除这个问题27.为了减少由于类器官异质性导致的批量效应，我们建议每次提取至?…
開示
The authors have nothing to disclose.
Acknowledgements
我们感谢 Valeria Fernandez-Vallone 提供 Miltenyi 神经解离套件的原始说明。我们还要感谢 Max Delbrueck Centrum 的基因组学技术平台提供 NP40 裂解缓冲液的配方和建立此方案的宝贵建议。我们还要感谢玛格丽塔·赫尔佐格（Margareta Herzog）和亚历山德拉·切尔尼切夫（Alexandra Tschernycheff）对实验室组织的支持。
Materials
| 1,4-DITHIO-DL-THREIT-LSG., F. D. MOL.-BIOL., ~1 M IN H2O (DTT) | Sigma | 43816-10ML | |
| 1.5 ml DNA low binding tubes | VWR | 525-0130 | microcentrifuge tube |
| 10x Cellranger pipeline | analysis pipline | ||
| 15 ml Falcon | Falcon | Centrifuge tube | |
| 2-Mercaptoethanol (BME) | Life Technologies | 21985023 | |
| 50 ml Falcon | Falcon | Centrifuge tube | |
| A83-01 | Bio Technologies | 379762 | |
| Antibiotic/Antimycotic Solution (100X) | Life Technologies | 15240062 | |
| B-27 Plus Supplement | Life Technologies | 17504044 | |
| B-27 Supplement without vitamin A | Life Technologies | 12587010 | |
| Bovine serum albumin, fatty acid free (BSA) | Sigma Aldrich | A8806-5G | |
| cAMP | Biogems | 6099240 | |
| cAMP | Biogems | 6099240 | |
| C-CHIP NEUBAUER IMPROVED | VWR | DHC-N01 | |
| Cell strainer 40 µm | Neolab | 352340 | |
| Cell strainer 70 µm (white) Nylon | Sigma | CLS431751-50EA | |
| Chromium Controller & Next GEM Accessory Kit | 10X Genomics | 1000204 | |
| Chromium Next GEM Chip G Single Cell Kit, 16 rxns | 10X Genomics | 1000127 | |
| Chromium Next GEM Single Cell 3' Kit v3.1 | 10X Genomics | 1000268 | |
| Complete, EDTA-free Protease Inhibitor Cocktaill | Roche | 11873580001 | |
| DAPI | MERCK Chemicals | 0000001722 | |
| DMEM/F12 | Life Technologies | 11320074 | |
| Dounce tissue grinder set 2 mL complete | Sigma Aldrich | 10536355 | |
| Essential E8 Flex Medium | Life Technologies | A2858501 | |
| EVE Cell Counting Slides | VWR | EVS-050 ( 734-2676) | |
| Foetal bovine serum tetracycline free (FBS) | PAN Biotech | P30-3602 | |
| Geltrex LDEV-Free (coating) | Life Technologies | A1413302 | |
| gentleMACS | Miltenyi Biotec | dissociation maschine | |
| GlutaMAX supplements | Life Technologies | 35050038 | |
| Heparin sodium cell culture tested | Sigma | H3149-10KU | |
| human recombinant BDNF | StemCell Technologies | 78005.3 | |
| human recombinant GDNF | StemCell Technologies | 78058.3 | |
| Insulin Solution Human | Sigma Aldrich | I2643-25MG | |
| Knockout serum replacement | Life Technologies | 10828028 | |
| LDN193189 Hydrochloride 98% | Sigma Aldrich | 130-106-540 | |
| MEM non-essential amino acid (100x) | Sigma Aldrich | M7145-100ml | |
| MgCl2 Magnesium Chloride (1M) RNAse free | Thermo Scientific | AM9530G | |
| mTeSR Plus | StemCell Technologies | 100-0276 | stem cell medium |
| mTeSR1 | StemCell Technologies | 85850 | stem cell medium |
| N2 Supplement | StemCell Technologies | 17502048 | |
| Neural Tissue Dissociation Kit | Miltenyi Biotec B.V. & Co. KG | 130-092-628 | |
| Neurobasal Plus | Life Technologies | A3582901 | |
| NextSeq500 system | Illumina | Sequencer | |
| NP-40 Surfact-Amps Detergent Solution | Life Technologies | 28324 | |
| PBS Dulbecco’s | Invitrogen | 14190169 | |
| PenStrep (Penicillin – Streptomycin) | Life Technologies | 15140122 | |
| Percoll | Th. Geyer | 10668276 | |
| Pluronic (R) F-127 | Sigma Aldrich | P2443-1KG | |
| RiboLock RNase Inhibitor | Life Technologies | EO0382 | |
| Rock Inhibitor (Y-27632 dihydrochloride) SB | Biomol | Cay10005583-10 | |
| SB 431542 | Biogems | 3014193 | |
| Sodium chloride NaCl (5M), RNase-free-100 mL | Invitrogen | AM9760G | |
| StemFlex Medium | Thermo Scientific | A3349401 | stem cell medium |
| StemMACS iPS-Brew XF | Miltenyi Biotec | 130-104-368 | stem cell medium |
| TC-Platte 96 Well, round bottom | Sarstedt | 83.3925.500 | |
| TISSUi006-A | TissUse GmbH | https://hpscreg.eu/cell-line/TISSUi006-A | |
| Trypan Blue | T8154-20ml | Sigma | |
| TrypLE Express Enzyme, no phenol red | Life Technologies | 12604013 | Trypsin-based reagent |
| UltraPure 1M Tris-HCl Buffer, pH 7.5 | Life Technologies | 15567027 | |
| XAV939 | Enzo Life sciences | BML-WN100-0005 |
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