使用 scRNA-Seq 和 scATAC-Seq 对视网膜基因表达和染色质可及性进行多重分析
1Solomon H. Snyder Department of Neuroscience,Johns Hopkins University School of Medicine, 2Department of Psychiatry and Behavioral Science,The Johns Hopkins Hospital, 3Department of Ophthalmology,The Johns Hopkins Hospital, 4Department of Neurology,The Johns Hopkins Hospital, 5Institute for Cell Engineering,Johns Hopkins University School of Medicine, 6Kavli Neuroscience Discovery Institute,Johns Hopkins University School of Medicine
Summary
在这里，作者展示了MULTI-seq用于表型的效用，以及随后配对的scRNA-seq和scATAC-seq在表征视网膜转录组和染色质可及性图谱方面的效用。
Abstract
强大的下一代测序技术提供了强大而全面的分析，以研究视网膜基因调控网络在发育和疾病状态下如何发挥作用。单细胞RNA测序使我们能够在细胞水平上全面分析在视网膜发育和疾病中观察到的基因表达变化，而单细胞ATAC-Seq允许分析染色质可及性和转录因子结合，以相似的分辨率进行分析。这里描述了这些技术在发育中的视网膜中的使用，并演示了MULTI-Seq，其中单个样品用修饰的寡核苷酸 – 脂质复合物标记，使研究人员能够增加单个实验的范围并大大降低成本。
Introduction
了解基因如何影响细胞命运在询问疾病和胚胎进展等过程中起着关键作用。转录因子与其靶基因之间的复杂关系可以在基因调控网络中分组。越来越多的证据表明，这些基因调控网络处于疾病和进化谱系发展的中心1。虽然以前的技术（如qRT-PCR）专注于单个基因或一组基因，但高通量测序技术的应用允许分析完整的细胞转录组。
RNA-seq提供了对大规模转录组学2，3的一瞥。单细胞RNA测序（scRNA-seq）使研究人员不仅能够分析转录组，还可以将特定细胞类型与基因表达谱联系起来4。这是通过使用已知的基因标记5将单个细胞谱输入到分选算法中来实现的生物信息学。使用脂质标记索引测序（MULTI-seq）进行多重检测，在可收集的scRNA-Seq谱数量方面提供了前所未有的多样性6.这种基于脂质的技术与其他样品索引技术不同，例如依赖于表面抗原和高亲和抗体的存在而不是质膜整合7的细胞散列。现在不仅可以将基因表达谱谱分析为细胞类型，还可以将不同的实验组合到单个测序文库中，从而大大降低了单个scRNA-seq实验的成本6。scRNA-seq的成本对于用于分析许多不同基因型，条件或患者样本的表型实验似乎令人望而却步，但多重检测允许在单个文库中组合多达96个样本6。
通过scRNA-seq分析基因表达并不是唯一一种基于高通量测序的技术，它彻底改变了目前对分子机制如何决定细胞命运的理解。虽然了解细胞中存在哪些基因转录本可以识别细胞类型，但同样重要的是了解基因组组织如何调节发育和疾病进展。早期研究依赖于检测DNase介导的不与组蛋白结合的序列的切割，然后对产生的DNA片段进行测序以鉴定开放染色质的区域。相比之下，转座子可访问染色质测序（scATAC-seq）的单细胞测定允许研究人员用驯化的转座子探测DNA，以便在单核苷酸水平8上轻松谱分析开放染色质。这已经经历了与scRNA-seq类似的缩放，现在研究人员可以识别单个细胞类型并分析数千个单个基因组中的表型8。
scRNA-seq和scATAC-seq的配对使研究人员能够分析数千个细胞，以确定疾病模型和发育过程中的细胞群，基因组组织和基因调控网络9，10，11，12。在这里，作者概述了如何首先利用 MULTI-seq 来凝聚无数动物模型的表型，并采用配对的 scRNA-seq 和 scATAC-seq 来更好地了解这些动物模型中的染色质景观和调控网络。
Protocol
这些研究中的动物使用是使用约翰霍普金斯大学动物护理和使用委员会批准的协议进行的，符合ARREAT指南，并按照相关指南和法规进行。 1. 多级 培养基制备 制备并平衡卵粘液抑制剂，10mg卵粘液抑制剂和每mL厄尔平衡盐溶液（EBSS）10mg白蛋白，使用前30分钟13。在培养箱中与 95% O2：5% CO2 平衡。在此孵育步骤<sup…
Representative Results
该工作流程列出了使用单细胞测序研究发育表型和调节过程的策略。MULTI-seq 样品多重检测可实现初始低成本表型分析，同时配对采集和固定 scRNA-seq 和 scATAC-seq 样品有助于进行更深入的研究（图 1）。 MULTI-seq 条形码可实现多个样本的组合排序及其随后的计算反卷积。可以根据每个细胞的条形码表达来确定其原产地样品（图2A）。这…
Discussion
MULTI-seq 的强大功能源于来自多个实验条件或模型的数据的无缝集成，以及在成本和限制批次效应方面的巨大优势。利用 MULTI-seq 提供了前所未有的实验室表型深度。非遗传多重检测方法，如细胞散列或细胞核散列，通过使用条形码抗体7，19，20为多路复用样品打开了大门。然而，这依赖于识别细胞或其细胞核上表达的表面蛋白?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢约翰霍普金斯大学转录组学和深度测序核心的 Linda Orzolek 帮助对产生的文库进行测序，并感谢蒋立志进行 离体 视网膜外植体。
Materials
| 10 µL, 200 µL, 1000 µL pipette filter tips | |||
| 10% Tween 20 | Bio-Rad | 1662404 | |
| 100 µM Barcode Solution | Request from Gartner lab | https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&edit_requested =true |
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| 100% Ethanol | Millipore Sigma | E7023-500ML | |
| 100% Methanol | Millipore Sigma | 322415-100ML | |
| 10x Chip Holder | 10x Genomics | 1000195 | |
| 10x Chromium controller & Accessory Kit | 10x Genomics | PN-120263 | |
| 15mL Centrifuge Tube | Quality Biological | P886-229411 | |
| 40 µm FlowMi Cell Strainer | Bel-Art | H13680-0040 | |
| 50 µM Anchor Solution | Sigma or request from Gartner lab | https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&edit_requested =true |
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| 50 µM Co-Anchor Solution | Sigma or request from Gartner lab | https://docs.google.com/forms/d/1bAzXFEvDEJse_cMvSUe_yDaP rJpAau4IPx8m5pauj3w/viewform?ts=5c47a897&edit_requested =true |
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| 5200 Fragment Analyzer system | Agilent | M5310AA | |
| 70 um FlowMi cell strainer | Bel-Art | H13680-0070 | |
| Allegra X-12R Centrifuge | VWR | BK392302 | |
| Bovine Serum Albumin | Sigma-Aldrich | A9647 | |
| Chromium Next GEM Chip G | 10x Genomics | PN-1000120 | |
| Chromium Next GEM Chip H | 10x Genomics | PN-1000161 | |
| Chromium Next Gem Single Cell ATAC Reagent Kit v1.1 | 10x Genomics | PN-1000175 | |
| Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 | 10x Genomics | PN-1000121 | |
| Digitonin | Fisher Scientific | BN2006 | |
| Dissection microscope | Leica | ||
| DNA LoBind Tubes, 1.5 mL | Eppendorf | 22431021 | |
| Dry Ice | |||
| EVA Foam Ice Pan | Tequipment | 04393-54 | |
| FA 12-Capillary Array Short, 33 cm | Agilent | A2300-1250-3355 | |
| Fisherbrand Isotemp Water Bath | Fisher Scientific | 15-460-20Q | |
| Forma CO2 Water Jacketed Incubator | ThermoFisher Scientific | 3110 | |
| Glycerol 50% Aqueous solution | Ricca Chemical Company | 3290-32 | |
| Hausser Scientific Bright-Line Counting Chamber | Fisher Scientific | 02-671-51B | |
| Illumina NextSeq or NovaSeq | Illumina | ||
| Kapa Hifi Hotstart ReadyMix | HiFi | 7958927001 | |
| Low TE Buffer | Quality Biological | 351-324-721 | |
| Magnesium Chloride Solution 1 M | Sigma-Aldrich | M1028 | |
| Magnetic Separator Rack for 1.5 mL tubes | Millipore Sigma | 20-400 | |
| Magnetic Separator Rack for 200 µL tubes | 10x Genomics | NC1469069 | |
| MULTI-seq Primer | Sigma or IDT | See sequence list | |
| MyFuge Mini Centrifuge | Benchmark Scientific | C1008 | |
| Nonidet P40 Substitute | Sigma-Aldrich | 74385 | |
| Nuclease-free water | Fisher Scientific | AM9937 | |
| P2, P10, P20, P200, P1000 micropipettes | Eppendorf | ||
| Papain Dissociation System | Worthington Biochemical Corporation | LK003150 | |
| PBS pH 7.4 (1X) | Fisher Scientific | 10010-023 | |
| Qiagen Buffer EB | Qiagen | 19086 | |
| Refridgerated Centrifuge 5424 R | Eppendorf | 2231000655 | |
| RNase-free Disposable Pellet Pestles | Fisher Scientific | 12-141-368 | |
| RNasin Plus RNase Inhibitor | Promega | N2615 | |
| RPI primer | Sigma or IDT | See sequence list | |
| Single Index Kit N, Set A | 10x Genomics | PN-1000212 | |
| Single Index Kit T Set A | 10x Genomics | PN-1000213 | |
| Sodium Chloride Solution 5 M | Sigma-Aldrich | 59222C | |
| SPRIselect Reagent Kit | Beckman Coulter | B23318 | |
| Standard Disposable Transfer Pipettes | Fisher Scientific | 13-711-7M | |
| TempAssure PCR 8-tube strip | USA Scientific | 1402-4700 | |
| Trizma Hydrochloride Solution, pH 7.4 | Sigma-Aldrich | T2194 | |
| Trypan Blue Solution, 0.4% (w/v) | Corning | 25-900-CI | |
| Universal I5 primer | Sigma or IDT | See sequence list | |
| Veriti Thermal Cycler | Applied Biosystems | 4375786 | |
| Vortex Mixer | VWR | 10153-838 |
Riferimenti
- Hoang, T., et al. Gene regulatory networks controlling vertebrate retinal regeneration. Science. 370, (2020).
- Nagalakshmi, U., et al. The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science. 320, 1344-1349 (2008).
- Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature Methods. 5, 621-628 (2008).
- Hwang, B., Lee, J. H., Bang, D. Single-cell RNA sequencing technologies and bioinformatics pipelines. Experimental & Molecular Medicine. 50, 96 (2018).
- Butler, A., Hoffman, P., Smibert, P., Papalexi, E., Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nature Biotechnology. 36, 411-420 (2018).
- McGinnis, C. S., et al. MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices. Nature Methods. 16, 619-626 (2019).
- Stoeckius, M., et al. Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biology. 19, 224 (2018).
- Chen, X., Miragaia, R. J., Natarajan, K. N., Teichmann, S. A. A rapid and robust method for single cell chromatin accessibility profiling. Nature Communications. 9, 5345 (2018).
- Hoang, T., et al. Gene regulatory networks controlling vertebrate retinal regeneration. Science. , 8598 (2020).
- Clark, B. S., et al. Single-Cell RNA-Seq Analysis of Retinal Development Identifies NFI Factors as Regulating Mitotic Exit and Late-Born Cell Specification. Neuron. 102, 1111-1126 (2019).
- Zheng, Y., et al. A human circulating immune cell landscape in aging and COVID-19. Protein Cell. 11, 740-770 (2020).
- Satpathy, A. T., et al. Massively parallel single-cell chromatin landscapes of human immune cell development and intratumoral T cell exhaustion. Nature Biotechnology. 37, 925-936 (2019).
- Worthington Biochemical Corporation. . Worthington Biochemical Corporation. Papain Dissociation System. , (2020).
- 10x Genomics. . Chromium Single Cell 3′ Reagent Kits v3 User Guide. , (2020).
- Agilent. . DNF-468 HS Genomic DNA 50 kb Kit Quick Guide for Fragment Analyzer Systems. , (2015).
- ThermoFisher Scientific. Qubit dsDNA HS Assay Kits. ThermoFisher Scientific. , (2015).
- 10x Genomics. . Chromium Single Cell ATAC Reagent Kits User Guide (v1.1 Chemistry). , (2020).
- Weir, K., Kim, D. W., Blackshaw, S. Regulation of retinal neurogenesis by somatostatin signaling. bioRxiv. , (2020).
- Stoeckius, M., et al. Simultaneous epitope and transcriptome measurement in single cells. Nature Methods. 14, 865-868 (2017).
- Gaublomme, J. T., et al. Nuclei multiplexing with barcoded antibodies for single-nucleus genomics. Nature Communications. 10, 2907 (2019).
- Ma, S., et al. Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin. Cell. , (2020).
- Buenrostro, J. D., et al. Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation. Cell. 173, 1535-1548 (2018).
- Pliner, H. A., et al. Cicero Predicts cis-Regulatory DNA Interactions from Single-Cell Chromatin Accessibility Data. Molecular Cell. 71, 858-871 (2018).
- Granja, J. M., et al. ArchR: An integrative and scalable software package for single-cell chromatin accessibility analysis. BioRxiv. , (2020).
- Stuart, T., et al. Comprehensive Integration of Single-Cell Data. Cell. 177, 1888-1902 (2019).
- METABRIC Group. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 486, 346-352 (2012).
- Izadi, F. Differential Connectivity in Colorectal Cancer Gene Expression Network. Iranian Biomedical Journal. 23, 34-46 (2019).
Citazione di questo articolo
Weir, K., Leavey, P., Santiago, C., Blackshaw, S. Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq. J. Vis. Exp. (169), e62239, doi:10.3791/62239 (2021).