小鼠胚胎细胞的多路复用单细胞mRNA测序分析
概要
在这里，我们提出了一种多路复用单细胞mRNA测序方法，用于分析小鼠胚胎组织中的基因表达。基于滴滴的单细胞mRNA测序（scRNA-Seq）方法与多路复用策略相结合，可以同时分析来自多个样品的单个细胞，从而显著降低试剂成本并最大限度地减少实验批次效应。
Abstract
单细胞mRNA测序在过去几年中取得了显著进展，已成为发育生物学领域的重要工具。它已成功用于识别稀有细胞群、发现新的标记基因以及解码空间和时间发育信息。单细胞方法也在过去两到三年中从基于微流体的Fluidigm C1技术发展到基于液滴的解决方案。在这里，我们以心脏为例，演示如何使用基于滴的scRNA-Seq方法分析小鼠胚胎组织细胞。此外，我们还将两种策略集成到工作流中，以在单个实验中分析多个样本。使用一种集成方法，我们同时从 8 个心脏样本中剖和分析了 9，000 多个细胞。这些方法将有价值的发育生物学领域，提供一个具有成本效益的方法，同时分析来自不同遗传背景，发育阶段或解剖位置的单个细胞。
Introduction
在胚胎发育期间，每个细胞的转录特征因细胞群而异。虽然单分子原位杂交可用于可视化少量基因1的表达，但单细胞mRNA测序（scRNA-Seq）提供了一种无偏见的方法来说明单个细胞中基因的全基因组表达模式。自2009年首次出版以来，scRNA-Seq已应用于研究多个发育阶段的多个组织，在近年3，4，5。此外，由于人类细胞图集最近启动了以发育为重点的项目，预计在不久的将来将生成更多来自人类胚胎组织的单细胞数据。
心脏作为发育的第一器官在胚胎发育中起着至关重要的作用。心脏由多种细胞类型组成，每种细胞类型的发育在时间和空间上受到严格调控。在过去几年中，心脏细胞在早期发育阶段的起源和细胞系一直以6为特征，这为了解先天性心脏病发病机制以及开发更先进的方法刺激心肌细胞再生提供了巨大的有用的导航工具。
scRNA-Seq在最近几年经历了迅速的扩张，8、9、10。随着新开发的方法，单细胞实验的设计和分析已经变得更加可实现11，12，13，14。这里介绍的方法是基于液滴溶液（见材料表）15，16的商业程序。该方法在微流体控制系统的控制下，在油水乳液液滴中捕获电池和一组唯一条形码的珠子。细胞加载到液滴的速率极低，因此大多数液滴乳液只含有一个细胞17。该程序的巧妙设计来自单细胞分离成滴乳液，与条形码同时发生，这使得使用RNA-Seq在异质种群上对单个细胞进行并行分析。
多路复用策略的整合是传统单单元工作流程13、14的重要补充之一。这种添加在丢弃细胞双子项，降低实验成本，并消除批处理效应18，19非常有用。基于脂质的条形码策略和基于抗体的条形码策略（见材料表）是两种最常用的多路复用方法。特定条形码用于在这两种方法中标记每个样本，然后混合标记的样本以进行单个单元捕获、库制备和排序。之后，通过分析条形码序列（图1）19，可以分离集合测序数据。但是，这两种方法之间存在显著差异。基于脂质的条形码策略基于脂质修饰寡核苷酸，尚未发现任何细胞类型偏好。而基于抗体的条形码策略只能检测出表达抗原蛋白的细胞19、20。此外，染色脂质大约需要10分钟，而染色抗体需要40分钟（图1）。此外，脂质修饰寡核苷酸比抗体结合的寡核苷酸便宜，但在撰写本文时没有市售。最后，基于脂质的策略可以在一个实验中多路复用96个样本，但基于抗体的策略目前只能多路复用12个样本。
在单个实验中，建议多路复用的细胞数应低于2.5 x 104，否则，将导致高百分比的细胞双子数和潜在的环境mRNA污染。通过多路复用策略，单个细胞捕获、cDNA生成和多个样品的库制备成本将降低到一个样本的成本，但测序成本将保持不变。
Protocol
动物程序符合匹兹堡大学机构动物护理和使用委员会（IACUC）。 1. 小鼠胚胎心脏解剖和单细胞悬浮准备 注：此步骤可能需要几个小时，具体取决于要解剖的胚胎数量。 为了获得E18.5胚胎心脏，通过CO2给给使怀孕的CD1小鼠安乐死。使用剃须刀去除腹部区域不需要的头发，用70%乙醇对皮肤进行消毒。 使用消毒剪刀切割腹部皮肤，仔细?…
Representative Results
在这项研究中，我们以小鼠胚胎心脏为例，展示如何同时进行多路复用单细胞mRNA测序，以同时处理来自器官不同部位的不同样本。E18.5 CD1 小鼠心脏被分离并解剖到左心房 （LA）、右心房 （RA）、左心室 （LV） 和右心室 （RV）。然后，心房和心室细胞使用基于脂质的条形码程序独立地进行条形码处理，并在 GEM 生成和逆转录前混合在一起。原理图概述如图1所?…
Discussion
在这项研究中，我们演示了分析单细胞转录配置文件的协议。我们还在scRNA-Seq工作流程中提供了两种可选方法，用于多路复用样品。这两种方法已证明在各种实验室是可行的，并提供了解决方案，运行一个经济高效和无批次效果的单细胞实验18，26。
在通过协议时，应仔细执行几个步骤。理想的单细胞悬浮液应具有>90%的活细胞，细?…
開示
The authors have nothing to disclose.
Acknowledgements
我们感谢Zev J. Gartner博士实验室的大卫·帕特森和克里斯托弗·麦金尼斯，他们提供了基于脂质的条形码试剂，并就实验步骤和数据分析提出了建议。这项工作是由国家卫生研究院（HL13347202）创立的。
Materials
| 10% Tween-20 | Bio-Rad | 1610781 | |
| 10x Chip Holder | 10x Genomics | 120252 330019 | |
| 10x Chromium Controller | 10x Genomics | 120223 | |
| 10x Magnetic Separator | 10x Genomics | 120250 230003 | |
| 10x Vortex Adapter | 10x Genomics | 330002, 120251 | |
| 10x Vortex Clip | 10x Genomics | 120253 230002 | |
| 4200 TapeStation System | Agilent | G2991AA | |
| Agilent High Sensitivity DNA Kit | Agilent | 5067-4626 | University of Pittsburgh Health Sciences Sequencing Core |
| Barcode Oligo | Integrated DNA Technologies | Single-stranded DNA | 25 nmol |
| Buffer EB | Qiagen | 19086 | |
| CD1 mice | Chales River | Strain Code 022 | ordered pregnant mice |
| Centrifuge 5424R | Appendorf | 2231000214 | |
| Chromium Chip B Single Cell Kit, 48 rxns | 10x Genomics | 1000073 | Store at ambient temperature |
| Chromium i7 Multiplex Kit, 96 rxns | 10x Genomics | 120262 | Store at -20 °C |
| Chromium Single Cell 3' GEM Kit v3,4 rxns | 10x Genomics | 1000094 | Store at -20 °C |
| Chromium Single Cell 3' Library Kit v3 | 10x Genomics | 1000095 | Store at -20 °C |
| Chromium Single Cell 3' v3 Gel Beads | 10x Genomics | 2000059 | Store at -80 °C |
| Collagenase A | Sigma/Millipore | 10103578001 | Store powder at 4 °C, store at -20 °C after it dissolves |
| Collagenase B | Sigma/Millipore | 11088807001 | Store powder at 4 °C, store at -20 °C after it dissolves |
| D1000 ScreenTape | Agilent | 5067-5582 | University of Pittsburgh Health Sciences Sequencing Core |
| DNA LoBind Tube Microcentrifuge Tube, 1.5 mL | Eppendorf | 022431021 | |
| DNA LoBind Tube Microcentrifuge Tube, 2.0 mL | Eppendorf | 022431048 | |
| Dynabeads MyOne SILANE | 10x Genomics | 2000048 | Store at 4 °C, used in Beads Cleanup Mix (Table 1) |
| DynaMag-2 Magnet | Theromo Scientific | 12321D | |
| Ethanol, Pure (200 Proof, anhydrous) | Sigma | E7023-500mL | |
| Falcon 15mL High Clarity PP Centrifuge Tube | Corning Cellgro | 14-959-70C | |
| Falcon 50mL High Clarity PP Centrifuge Tube | Corning Cellgro | 14-959-49A | |
| Fetal Bovine Serum, qualified, United States | Fisher Scientific | 26140079 | Store at -20 °C |
| Finnpipette F1 Multichannel Pipettes, 10-100μl | Theromo Scientific | 4661020N | |
| Finnpipette F1 Multichannel Pipettes, 1-10μl | Theromo Scientific | 4661000N | |
| Flowmi Cell Strainer | Sigma | BAH136800040 | Porosity 40 μm, for 1000 uL Pipette Tips, pack of 50 each |
| Glycerin (Glycerol), 50% (v/v) | Ricca Chemical Company | 3290-32 | |
| HBSS, no calcium, no magnesium | Thermo Fisher Scientific | 14170112 | |
| Human TruStain FcX (Fc Receptor Blocking Solution) | BioLegend | 422301 | Add 5 µl of Human TruStain FcX per million cells in 100 µl staining volume |
| Isopropanol (IPA) | Fisher Scientific | A464-4 | |
| Kapa HiFi HotStart ReadyMix (2X) | Fisher Scientific | NC0295239 | Store at -20 °C, used in Lipid-tagged barcode library mix (Table 1) |
| Lipid Barcode Primer (Multi-seq Primer) | Integrated DNA Technologies | Single-stranded DNA | 100 nmol |
| Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) | Thermo Fisher Scientific | 12090-015 | |
| MasterCycler Pro | Eppendorf | 950W | |
| Nuclease-Free Water (Ambion) | Thermo Fisher Scientific | AM9937 | |
| PCR Tubes 0.2 ml 8-tube strips | Eppendorf | 951010022 | |
| Phosphate-Buffered Saline (PBS) 1X without calcium & magnesium | Corning Cellgro | 21-040-CV | |
| Phosphate-Buffered Saline (PBS) with 10% Bovine Albumin (alternative to Thermo Fisher product) | Sigma-Aldrich | SRE0036 | |
| Pipet 4-pack (0.1–2.5μL, 0.5-10μL, 10–100μL, 100–1,000μL variable-volume pipettes | Fisher Scientific | 05-403-151 | |
| Selection reagent (SPRIselect Reagent Kit) | Beckman Coulter | B23318 (60ml) | |
| Template Switch Oligo | 10x Genomics | 3000228 | Store at -20 °C, used in Master Mix (Table 1) |
| The antibody based barcoding strategy is also known as Cell Hashing | |||
| The cell browser is Loup Cell Browser | 10x Genomics | https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser | |
| The commercial available analysis pipline in step 8.1 is Cell Ranger | 10x Genomics | https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger | |
| The lipid based barcoding strategy is also known as MULTI-seq | |||
| The well maintained R platform is Seurat V3 | satijalab | https://satijalab.org/seurat/ | |
| TipOne RPT 0.1-10/20 ul XL ultra low retention filter pipet tip | USA Scientific | 1180-3710 | |
| TipOne RPT 1000 ul XL ultra low retention filter pipet tip | USA Scientific | 1182-1730 | |
| TipOne RPT 200 ul ultra low retention filter pipet tip | USA Scientific | 1180-8710 | |
| TotalSeq-A0301 anti-mouse Hashtag 1 Antibody | BioLegend | 155801 | 0.1 – 1.0 µg of antibody in 100 µl of staining buffer for every 1 million cells |
| TotalSeq-A0302 anti-mouse Hashtag 2 Antibody | BioLegend | 155803 | 0.1 – 1.0 µg of antibody in 100 µl of staining buffer for every 1 million cells |
| TotalSeq-A0302 anti-mouse Hashtag 3 Antibody | BioLegend | 155805 | 0.1 – 1.0 µg of antibody in 100 µl of staining buffer for every 1 million cells |
| TrueSeq RPI primer | Integrated DNA Technologies | Single-stranded DNA | 100 nmol, used in Lipid-tagged barcode library mix (Table 1) |
| Trypan Blue Solution, 0.4% | Fisher Scientific | 15250061 | |
| Trypsin-EDTA (0.25%), phenol red | Fisher Scientific | 25200-056 | |
| Universal I5 | Integrated DNA Technologies | Single-stranded DNA | 100 nmol |
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