Name: multiplexed single cell mrna sequencing analysis of mouse embryonic cells
Uploaded: 2020-01-07T14:00:00
Description: Watch this Scientific Journal Video about Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells at JoVE.com

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。

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。
<ol><li>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。</li>
	<li>使用消毒剪刀切割腹部皮肤，仔细?...

<ol>
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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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Tween-20</td>
			<td>Bio-Rad</td>
			<td>1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Chip Holder</td>
			<td>10x Genomics</td>
			<td>120252 330019</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Chromium Controller</td>
			<td>10x Genomics</td>
			<td>120223</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Magnetic Separator</td>
			<td>10x Genomics</td>
			<td>120250 230003</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Vortex Adapter</td>
			<td>10x Genomics</td>
			<td>330002, 120251</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Vortex Clip</td>
			<td>10x Genomics</td>
			<td>120253 230002</td>
			<td></td>
		</tr>
		<tr>
			<td>4200 TapeStation System</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td>University of Pittsburgh Health Sciences Sequencing Core</td>
		</tr>
		<tr>
			<td>Barcode Oligo</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>25 nmol</td>
		</tr>
		<tr>
			<td>Buffer EB</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1 mice</td>
			<td>Chales River</td>
			<td>Strain Code 022</td>
			<td>ordered pregnant mice</td>
		</tr>
		<tr>
			<td>Centrifuge 5424R</td>
			<td>Appendorf</td>
			<td>2231000214</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromium Chip B Single Cell Kit, 48 rxns</td>
			<td>10x Genomics</td>
			<td>1000073</td>
			<td>Store at ambient temperature</td>
		</tr>
		<tr>
			<td>Chromium i7 Multiplex Kit, 96 rxns</td>
			<td>10x Genomics</td>
			<td>120262</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; GEM Kit v3,4 rxns</td>
			<td>10x Genomics</td>
			<td>1000094</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; Library Kit v3</td>
			<td>10x Genomics</td>
			<td>1000095</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; v3 Gel Beads</td>
			<td>10x Genomics</td>
			<td>2000059</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Sigma/Millipore</td>
			<td>10103578001</td>
			<td>Store powder at 4 &#176;C, store at -20 &#176;C after it dissolves</td>
		</tr>
		<tr>
			<td>Collagenase B</td>
			<td>Sigma/Millipore</td>
			<td>11088807001</td>
			<td>Store powder at 4 &#176;C, store at -20 &#176;C after it dissolves</td>
		</tr>
		<tr>
			<td>D1000 ScreenTape</td>
			<td>Agilent</td>
			<td>5067-5582</td>
			<td>University of Pittsburgh Health Sciences Sequencing Core</td>
		</tr>
		<tr>
			<td>DNA LoBind Tube Microcentrifuge Tube, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>022431021</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind Tube Microcentrifuge Tube, 2.0 mL</td>
			<td>Eppendorf</td>
			<td>022431048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne SILANE</td>
			<td>10x Genomics</td>
			<td>2000048</td>
			<td>Store at 4 &#176;C, used in Beads Cleanup Mix (Table 1)</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Theromo Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, Pure (200 Proof, anhydrous)</td>
			<td>Sigma</td>
			<td>E7023-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15mL High Clarity PP Centrifuge Tube</td>
			<td>Corning Cellgro</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50mL High Clarity PP Centrifuge Tube</td>
			<td>Corning Cellgro</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, United States</td>
			<td>Fisher Scientific</td>
			<td>26140079</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Finnpipette F1 Multichannel Pipettes, 10-100&#956;l</td>
			<td>Theromo Scientific</td>
			<td>4661020N</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F1 Multichannel Pipettes, 1-10&#956;l</td>
			<td>Theromo Scientific</td>
			<td>4661000N</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowmi Cell Strainer</td>
			<td>Sigma</td>
			<td>BAH136800040</td>
			<td>Porosity 40 &#956;m, for 1000 uL Pipette Tips, pack of 50 each</td>
		</tr>
		<tr>
			<td>Glycerin (Glycerol), 50% (v/v)</td>
			<td>Ricca Chemical Company</td>
			<td>3290-32</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14170112</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX (Fc Receptor Blocking Solution)</td>
			<td>BioLegend</td>
			<td>422301</td>
			<td>Add 5 &#181;l of Human TruStain FcX per million cells in 100 &#181;l staining volume</td>
		</tr>
		<tr>
			<td>Isopropanol (IPA)</td>
			<td>Fisher Scientific</td>
			<td>A464-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa HiFi HotStart ReadyMix (2X)</td>
			<td>Fisher Scientific</td>
			<td>NC0295239</td>
			<td>Store at -20 &#176;C, used in Lipid-tagged barcode library mix (Table 1)</td>
		</tr>
		<tr>
			<td>Lipid Barcode Primer (Multi-seq Primer)</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>100 nmol</td>
		</tr>
		<tr>
			<td>Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090-015</td>
			<td></td>
		</tr>
		<tr>
			<td>MasterCycler Pro</td>
			<td>Eppendorf</td>
			<td>950W</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water (Ambion)</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Tubes 0.2 ml 8-tube strips</td>
			<td>Eppendorf</td>
			<td>951010022</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) 1X without calcium &#38; magnesium</td>
			<td>Corning Cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) with 10% Bovine Albumin (alternative to Thermo Fisher product)</td>
			<td>Sigma-Aldrich</td>
			<td>SRE0036</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 4-pack (0.1&#8211;2.5&#956;L, 0.5-10&#956;L, 10&#8211;100&#956;L, 100&#8211;1,000&#956;L variable-volume pipettes</td>
			<td>Fisher Scientific</td>
			<td>05-403-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection reagent (SPRIselect Reagent Kit)</td>
			<td>Beckman Coulter</td>
			<td>B23318 (60ml)</td>
			<td></td>
		</tr>
		<tr>
			<td>Template Switch Oligo</td>
			<td>10x Genomics</td>
			<td>3000228</td>
			<td>Store at -20 &#176;C, used in Master Mix (Table 1)</td>
		</tr>
		<tr>
			<td>The antibody based barcoding strategy is also known as Cell Hashing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The cell browser is Loup Cell Browser</td>
			<td>10x Genomics</td>
			<td></td>
			<td><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser" target="_blank">https://support.10xgenomics.com/single-cell-gene-expression/software/visualization/latest/what-is-loupe-cell-browser</a></td>
		</tr>
		<tr>
			<td>The commercial available analysis pipline in step 8.1 is Cell Ranger</td>
			<td>10x Genomics</td>
			<td></td>
			<td><a href="https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger" target="_blank">https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger</a></td>
		</tr>
		<tr>
			<td>The lipid based barcoding strategy is also known as MULTI-seq</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The well maintained R platform is Seurat V3</td>
			<td>satijalab</td>
			<td></td>
			<td><a href="https://satijalab.org/seurat/" target="_blank">https://satijalab.org/seurat/</a></td>
		</tr>
		<tr>
			<td>TipOne RPT 0.1-10/20 ul XL ultra low retention filter pipet tip</td>
			<td>USA Scientific</td>
			<td>1180-3710</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne RPT 1000 ul XL ultra low retention filter pipet tip</td>
			<td>USA Scientific</td>
			<td>1182-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne RPT 200 ul ultra low retention filter pipet tip</td>
			<td>USA Scientific</td>
			<td>1180-8710</td>
			<td></td>
		</tr>
		<tr>
			<td>TotalSeq-A0301 anti-mouse Hashtag 1 Antibody</td>
			<td>BioLegend</td>
			<td>155801</td>
			<td>0.1 - 1.0 &#181;g of antibody in 100 &#181;l of staining buffer for every 1 million cells</td>
		</tr>
		<tr>
			<td>TotalSeq-A0302 anti-mouse Hashtag 2 Antibody</td>
			<td>BioLegend</td>
			<td>155803</td>
			<td>0.1 - 1.0 &#181;g of antibody in 100 &#181;l of staining buffer for every 1 million cells</td>
		</tr>
		<tr>
			<td>TotalSeq-A0302 anti-mouse Hashtag 3 Antibody</td>
			<td>BioLegend</td>
			<td>155805</td>
			<td>0.1 - 1.0 &#181;g of antibody in 100 &#181;l of staining buffer for every 1 million cells</td>
		</tr>
		<tr>
			<td>TrueSeq RPI primer</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>100 nmol, used in Lipid-tagged barcode library mix (Table 1)</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Fisher Scientific</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal I5</td>
			<td>Integrated DNA Technologies</td>
			<td>Single-stranded DNA</td>
			<td>100 nmol</td>
		</tr>
	</tbody>
</table>

multiplexed single cell mrna sequencing analysis of mouse embryonic cells

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。

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?...

Watch this Scientific Journal Video about Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells at JoVE.com

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

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。

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

单细胞mRNA测序被广泛用于了解单个细胞级别的生物过程。多路复用策略的出现进一步扩大了其放大，允许在单个实验中分析多个样本，从而大大降低了成本并避免了批处理效应。演示这个程序的将是魏峰，博士后，和安德鲁·普日辛达，一个技术员从我实验室。计数后，将从胚胎18.5个心脏室分离出的细胞分成5倍10至第5个等分，每次洗涤用新鲜PBS洗两次细胞。每样品将颗粒重新加入 180 微升 PBS 中，并通过温和混合将 20 微升锚状条形码库存溶液添加到每个管中。在冰上孵育5分钟后，将20微升共锚条形码储存溶液加入每个管，轻轻混合，在冰上再孵育5分钟，然后用1毫升的冷PBS洗涤细胞三次，每次洗涤1%牛血清白蛋白，然后通过每管一个40微米细胞过滤器将样品过滤到新管中。计数后，将芯片 B 组装到芯片支架中，然后在第一排未使用的油井中加入 75 微升 50%甘油溶液，在第二排未使用的油井中加入 40 微升，在第三排未使用的油井中加入 280 微升。请注意，此处使用训练芯片来演示该过程。通过温和的移液将适当的细胞悬浮液和无核酸酶水添加到主混合物中。将生成的细胞溶液的 75 微升添加到样品井的底部中心，第一行没有气泡。Vortex 凝胶珠30秒，然后缓慢地将40微升珠添加到凝胶珠的底部中心，在第二排没有气泡。在第三排分区油井的侧壁上加入 280 微升的分区油。将垫片连接到芯片上，无需按下垫片，且垫片保持水平，以避免弄湿垫片。在铬控制器中用垫片加载组装的芯片，并运行铬单单元B程序。请注意，屏幕显示训练芯片的铬训练，但对于实际实验，屏幕将显示铬单细胞B。将盖子折回，以 45 度角露出油井，并检查液位，确保不存在堵塞。从回收液的最低点缓慢吸出乳液中的100微升凝胶珠，并检查珠子的均匀性。将乳化珠子从新的 PCR 管的墙壁上分配到冰上，然后将管放在热循环器中进行逆转录。要准备cDNA进行扩增，在室温下将125微升的回收剂加入样品中。不应观察到不透明的液体，避免移液或涡流。等待 60 秒，然后慢慢从管底取出回收剂，并将 200 微升涡珠清洁混合物添加到样品中。在室温下孵育10分钟之前，先将混合物移液10次。接下来，将样品放在高位置的磁铁上。当溶液清除时，去除上清液，并在颗粒中加入200微升80%乙醇。30 秒后，取出乙醇并重复洗涤两次。上次洗涤后，将样品短暂离车，将管子放在磁体上的低位置，使样品风干不到两分钟。当样品干燥后，从磁铁上取出管子，并加入35.5微升新鲜制备的洗脱溶液。在室温下孵育两分钟后，将样品放在磁铁的高位置，直到溶液清除，然后再将样品的35微升转移到新的管条上。对于使用脂质条形码策略的cDNA扩增，在短暂离心前，在35微升cDNA样品中加入扩增反应混合物，并进行彻底混合。在旋转结束时，按照适当的 cDNA 扩增过程在热循环器中孵育样品。将 120 微升精选试剂和 100 微升超纯水添加到 100 微升样品中，并移液产生的 0.6 倍精选试剂 15 次。在室温下孵育五分钟后，将样品放在磁铁上，直到溶液变清。将上一液转移到 1.5 毫升低绑定管中，用于多路复用条码 cDNA 库结构。请记住，当上清器的内容组装条形码cDNA和基物内生cDNA时，请同时保存超自然物质和碱基。用80%乙醇清洁内源性cDNA，用40微升洗脱缓冲液洗脱DNA，然后对内源性cDNA进行质量控制检查，然后准备内源性转录库。在库建设前，应量化和限定cDNA浓度。构造的库，包括内生cDNA和条形码库，在测序之前也应该进行量化和限定。测序后，可以在每个单元中分析条形码表达式。例如，在此分析中观察到八组单细胞，它们唯一地表示一种类型的条形码，表示来自八个不同样本的细胞。此外，一些单元格不表示任何条形码，因此被定义为负细胞，而其他单元格表示两个不同的条形码表示双精度。利用单细胞，可以通过细胞类型注释、新颖和稀有细胞类型识别、解剖区比较分析、基因本体通路分析（如细胞周期相分离）来评估细胞异质性和分子调控。如果可以，在乳液中成像凝胶珠是有帮助的，因为图像会告诉你手术是否成功到现在。示例多路复用增加了设计实验的灵活性。看完这个演示，希望你们能更有信心开始自己的实验。

Research

JoVE Journal

Developmental Biology

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...