Name: a combinatorial single-cell approach to characterize the molecular and immunophenotypic heterogeneity of human stem and progenitor populations
Uploaded: 2018-10-25T12:30:00
Description: Watch this Scientific Journal Video about A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations at JoVE.com

这项工作得到了瑞典癌症协会、瑞典研究委员会、瑞典医学研究协会、瑞典儿童癌症基金会、拉格纳 Söderberg 基金会以及克努特和爱丽丝基金会的资助。

1. 裂解板的制备
<ol><li>使用 RNA/DNA 自由工作台, 准备足够的裂解缓冲液为96孔, 10% 额外, 通过混合390µL 核酸酶游离水, 17 µL 10% NP-40, 2.8 µL 10 毫米 dNTP, 10 µL 0.1 M DTT 和5.3 µL 核糖核酸酶抑制剂 (见材料表)。涡旋向下。</li>
	<li>将4µL 裂解缓冲液分布到96孔 PCR 板的每个井中, 并用胶膜密封板。在盘子底部旋下管子以收集液体。保持板在冰上直到细胞分类 (最大24小时)。</li>
</ol><p class="jove_tit...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Batch+effects+and+the+effective+design+of+single-cell+gene+expression+studies.">Batch effects and the effective design of single-cell gene expression studies.</a> Scientific Reports. 7, 39921 (2017).</li><li>Teles, J., Enver, T., Pina, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+PCR+profiling+of+gene+expression+in+hematopoiesis.">Single-cell PCR profiling of gene expression in hematopoiesis.</a> Methods in Molecular Biology. , 21-42 (2014).</li><li>Hayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+gene+profiling+of+planarian+stem+cells+using+fluorescent+activated+cell+sorting+and+its+&amp;#34;index+sorting&amp;#34;+function+for+stem+cell+research.">Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its &amp;#34;index sorting&amp;#34; function for stem cell research.</a> Development, Growth, &amp; Differentiation. 52 (1), 131-144 (2010).</li><li>Lang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCExV:+A+webtool+for+the+analysis+and+visualisation+of+single+cell+qRT-PCR+data.">SCExV: A webtool for the analysis and visualisation of single cell qRT-PCR data.</a> BMC Bioinformatics. 16 (1), 320 (2015).</li><li>de Klein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cellular+oncogene+is+translocated+to+the+Philadelphia+chromosome+in+chronic+myelocytic+leukaemia.">A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukaemia.</a> Nature. 300 (5894), 765-767 (1982).</li><li>. indexed-sorting Available from: <a target="_blank" href="https://github.com/FlowJo-LLC/indexed-sorting">https://github.com/FlowJo-LLC/indexed-sorting</a> (2016)</li><li>Warfvinge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+molecular+analysis+defines+therapy+response+and+immunophenotype+of+stem+cell+subpopulations+in+CML.">Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.</a> Blood. 129 (17), 2384-2394 (2017).</li><li>Nestorowa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+resolution+map+of+mouse+hematopoietic+stem+and+progenitor+cell+differentiation.">A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.</a> Blood. 128 (8), e20-e31 (2016).</li><li>Breton, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dendritic+cells+(DCs)+are+derived+from+distinct+circulating+precursors+that+are+precommitted+to+become+CD1c++or+CD141++DCs.">Human dendritic cells (DCs) are derived from distinct circulating precursors that are precommitted to become CD1c+ or CD141+ DCs.</a> The Journal of Experimental Medicine. 213 (13), 2861-2870 (2016).</li><li>Alberti-Servera, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+reveals+developmental+heterogeneity+among+early+lymphoid+progenitors.">Single-cell RNA sequencing reveals developmental heterogeneity among early lymphoid progenitors.</a> The EMBO Journal. 36 (24), 3619-3633 (2017).</li><li>Giustacchini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+uncovers+distinct+molecular+signatures+of+stem+cells+in+chronic+myeloid+leukemia.">Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.</a> Nature Medicine. 23, 692 (2017).</li><li>Hansmann, L., Han, A., Penter, L., Liedtke, M., Davis, M. 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近年来, 单细胞基因表达分析已成为定义不同细胞种群23的异质性的重要补充。RNA 测序技术的出现在理论上提供了测量细胞的整个转录组的可能性, 然而这些方法由于细胞到细胞测序深度和辍学的变化而变得复杂。单细胞的 qPCR 提供了对数以百计的关键基因的表达的灵敏和稳健的分析, 其中所有细胞的处理类似, 减少技术噪音。有限数量的成绩单的集中分析还允许简化分析, 而不会...

每个单独的血细胞被认为驻留在细胞的层次结构中, 其中干细胞形成了一系列日益承诺的中间祖细胞顶部的顶点, 最终分化成具有特定的最终效应细胞生物功能1。许多关于干细胞系统是如何组织的知识是在造血系统中产生的, 这主要是因为能够前瞻性地隔离高浓度的干细胞或各种祖细胞的不同造血种群2的排序。这允许许多这些人口被分析功能或分子, 主要通过基因表达分析3,4。然而, 当分析大体积种群的基因表达时, 细胞间的个体差异平均为5, 损失较大。因此, 如果细胞的小子集占该种群6的推断生物学功能, 那么无法检测异质细胞分数内的细胞对细胞的变化可能会混淆我们对关键生物过程的理解, 7。相反, 在单细胞分辨率的基因表达特征的研究提供了一种可能性, 描绘异质性和规避遮蔽影响从8个以上的单元格子集。
迄今已开发出许多单细胞基因表达分析的协议;每种方法都有自己的注意事项。最早的方法是 rna 荧光原位杂交 (rna-鱼), 它一次测量有限数量的转录, 但它是唯一的, 它允许调查 RNA 本地化9,11。早期的方法使用 PCR 和 qPCR 检测单个或很少的转录, 也开发了12。然而, 这些最近被取代了基于微流体的方法, 可以同时分析数以百计的每细胞在数以百计的细胞通过 qPCR 的表达, 从而允许高维异质性分析使用预先确定的基因面板10,13。最近, 基于 RNA 测序技术已广泛用于单细胞分析, 因为这些理论可以测量细胞的整个转录组, 从而为异质性分析10添加探索维度, 14. 复用的 qPCR 分析和单细胞 RNA 测序具有不同的特征, 因此使用这两种方法的基本原理取决于所问的问题以及目标种群中的细胞数量。当未知细胞或大种群被调查时, 每个细胞的高通量和低成本以及无偏的、探索性的单细胞 RNA 测序特性是可取的。然而, 单细胞 RNA 测序也偏向于更频繁地测序高丰富的转录, 而低丰度的成绩单容易辍学。这可能导致相当复杂的数据, 使高要求的生物信息学分析, 以揭示重要的分子信号, 往往微妙或隐藏在技术噪声15。因此, 对于特征良好的组织, 使用预先确定的底漆面板选择用于功能重要基因或分子标记的单细胞 qPCR 分析可以作为一种敏感、直接的方法来确定人口。然而, 应该指出的是, 与单细胞 RNA 序列相比, 单细胞的 qPCR 方法的每个细胞的成本通常更高。在这里, 我们描述了一种结合单细胞 rt-pcr 的方法 (从 Teles J. 16), 将指数排序17和生物信息学分析18 , 以同时表征种群内的分子和免疫表型异质性。
在这种方法中, 细胞种群的兴趣被染色, 单细胞通过直接在96孔 PCR 板的裂解缓冲液中进行排序。同时, 在流式排序过程中, 为每个单个单元记录一组附加的单元表面标记的表达式级别, 该方法称为索引排序。随后用微流控平台对裂解细胞材料进行扩增, 并用 rt-pcr 分析了选定基因组的基因表达。该策略可对已排序的单细胞进行分子分析, 同时表征每个细胞的细胞表面标记表达。通过直接将分子不同的细胞子集映射到索引排序标记的表达式, 可以将亚群链接到可用于潜在隔离的特定免疫表型。图 1中逐步概述了该方法。预先确定的基因面板进一步有助于更高分辨率的靶向基因表达, 因为它绕过不相关的丰富的基因的测量, 否则可能咬合微妙的基因表达信号。此外, 具体的靶向放大、一步反转转录和扩增允许对低表达转录物 (如转录因子或非聚仍然 rna) 进行稳健测量。重要的是, qPCR 方法允许测量融合蛋白的 mRNA, 这在调查某些恶性疾病时非常重要19。最后, 被调查的基因的重点数量, 低辍学率, 和有限的技术差异的细胞使这种方法容易分析相比, 更高的维度方法, 如单细胞 RNA-通过遵循该协议, 可以在三天内完成整个实验, 从分拣细胞到分析结果, 使之成为敏感、高通量单细胞基因表达分析的一种简单而快速的方法。

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		<tr>
			<td>CD14 PECY5</td>
			<td>eBioscience</td>
			<td>15-0149-42</td>
			<td>Clone: 61D3</td>
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			<td>CD16 PECY5</td>
			<td>Biolegend</td>
			<td>302010</td>
			<td>Clone: 3G8</td>
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			<td>CD56 PECY5</td>
			<td>Biolegend</td>
			<td>304608</td>
			<td>Clone: MEM-188</td>
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		<tr>
			<td>CD19 PECY5</td>
			<td>Biolegend</td>
			<td>302210</td>
			<td>Clone: HIB19</td>
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		<tr>
			<td>CD2 PECY5</td>
			<td>Biolegend</td>
			<td>300210</td>
			<td>Clone: RPA-2.10</td>
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			<td>CD3 PECY5</td>
			<td>Biolegend</td>
			<td>300310</td>
			<td>Clone: HIT3a</td>
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			<td>CD123 PECY5</td>
			<td>Biolegend</td>
			<td>306008</td>
			<td>Clone: 6H6</td>
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			<td>CD235A PECY5</td>
			<td>BD Pharma</td>
			<td>559944</td>
			<td>Clone GAR2</td>
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			<td>CD34 FITC</td>
			<td>Biolegend</td>
			<td>343604</td>
			<td>Clone: 561</td>
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			<td>CD38 APC</td>
			<td>Biolegend</td>
			<td>303510</td>
			<td>Clone: Hit2</td>
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			<td>CD90 PE</td>
			<td>Biolegend</td>
			<td>328110</td>
			<td>Clone: 5E10</td>
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			<td>CD45RA BV421</td>
			<td>BD bioscience</td>
			<td>560362</td>
			<td>Clone: HI100</td>
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			<td>CD49f Pecy7</td>
			<td>eBioscience</td>
			<td>25-0495-82</td>
			<td>Clone: eBioGOH3</td>
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			<td>FBS</td>
			<td>HyClone</td>
			<td>SV30160.3</td>
			<td></td>
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			<td>PBS</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
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			<td>96-well u-bottom Plate</td>
			<td>VWR</td>
			<td>10861-564</td>
			<td></td>
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			<td>SFEM</td>
			<td>Stem cell technologies</td>
			<td>9650</td>
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			<td>Penicillin streptomycin</td>
			<td>HyClone</td>
			<td>SV30010</td>
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			<td>TPO</td>
			<td>Peprotech</td>
			<td>300-18</td>
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			<td>SCF</td>
			<td>Peprotech</td>
			<td>300-07</td>
			<td></td>
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		<tr>
			<td>FLT3L</td>
			<td>Peprotech</td>
			<td>300-19</td>
			<td></td>
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		<tr>
			<td>Falcon Tube 15 mL</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td></td>
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		<tr>
			<td>Eppendorph tube</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>CST beads</td>
			<td>BD</td>
			<td>642412</td>
			<td></td>
		</tr>
		<tr>
			<td>Accudrop Beads</td>
			<td>BD</td>
			<td>345249</td>
			<td>6-&#181;m particles&#160;</td>
		</tr>
		<tr>
			<td>Adhesive film Clear</td>
			<td>Thermo scientific&#160;</td>
			<td>AB-1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive film Foil</td>
			<td>Thermo scientific&#160;</td>
			<td>AB-0626</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well PCR plate</td>
			<td>Axygen</td>
			<td>PCR-96M2-HS-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR 1.5 mL tube</td>
			<td>Axygen</td>
			<td>MCT-150-L-C</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 PCR cycler</td>
			<td>BioRad</td>
			<td>186-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP40</td>
			<td>Thermo scientific&#160;</td>
			<td>85124</td>
			<td></td>
		</tr>
		<tr>
			<td>10mM dNTP</td>
			<td>Takara</td>
			<td>4030</td>
			<td></td>
		</tr>
		<tr>
			<td>0.1M DTT</td>
			<td>Invitrogen</td>
			<td>P2325</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAsout</td>
			<td>Invitrogen</td>
			<td>10777-019</td>
			<td>RNAse inhibitor</td>
		</tr>
		<tr>
			<td>CellsDirect One-Step qRT-PCR Kit</td>
			<td>Invitrogen</td>
			<td>11753-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuclease free water</td>
			<td>Invitrogen</td>
			<td>11753-100</td>
			<td>from CellsDirect kit</td>
		</tr>
		<tr>
			<td>2X Reaction Mix</td>
			<td>Invitrogen</td>
			<td>11753-100</td>
			<td>from CellsDirect kit</td>
		</tr>
		<tr>
			<td>SuperScript III RT/Platinum Taq Mix</td>
			<td>Invitrogen</td>
			<td>11753-100</td>
			<td>from CellsDirect kit</td>
		</tr>
		<tr>
			<td>Platinum&#160;Taq&#160;DNA Polymerase</td>
			<td>Invitrogen</td>
			<td>10966026</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Cells-to-CT Control Kit</td>
			<td>Invitrogen</td>
			<td>4386995</td>
			<td></td>
		</tr>
		<tr>
			<td>Xeno RNA Control</td>
			<td>Invitrogen</td>
			<td>4386995</td>
			<td>From TaqMan Cells-to-CT Control Kit</td>
		</tr>
		<tr>
			<td>20X Xeno RNA Control Taqman Gene Expression Assay</td>
			<td>Invitrogen</td>
			<td>4386995</td>
			<td>From TaqMan Cells-to-CT Control Kit</td>
		</tr>
		<tr>
			<td>96.96 Sample/Loading Kit&#8212;10 IFCs</td>
			<td>Fluidigm</td>
			<td>BMK-M10-96.96</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Assay Loading Reagent</td>
			<td>Fluidigm</td>
			<td></td>
			<td>From 96.96 Sample/Loading Kit</td>
		</tr>
		<tr>
			<td>20X GE Sample Loading Reagent</td>
			<td>Fluidigm</td>
			<td></td>
			<td>From 96.96 Sample/Loading Kit</td>
		</tr>
		<tr>
			<td>Control line fluid&#160;</td>
			<td>Fluidigm</td>
			<td></td>
			<td>From 96.96 Sample/Loading Kit</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr>
			<td>BioMark HD</td>
			<td>Fluidigm</td>
			<td>BMKHD-BMKHD</td>
			<td></td>
		</tr>
		<tr>
			<td>96.96 Dynamic Array IFC</td>
			<td>Fluidigm</td>
			<td>BMK-M10-96.96GT</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>Microsoft</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo V10</td>
			<td>TreeStar</td>
			<td>TreeStar</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluidigm real time PCR analysis</td>
			<td>Fluidigm</td>
			<td>Fluidigm</td>
			<td></td>
		</tr>
		<tr>
			<td>CD179a.VPREB1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00356766_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>ACE</td>
			<td>Thermofisher scientific</td>
			<td>Hs00174179_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>AHR</td>
			<td>Thermofisher scientific</td>
			<td>Hs00169233_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>BCR_ABL.52</td>
			<td>Thermofisher scientific</td>
			<td>Hs03043652_ft</td>
			<td></td>
		</tr>
		<tr>
			<td>BCR_ABL41</td>
			<td>Thermofisher scientific</td>
			<td>Hs03024541_ft</td>
			<td></td>
		</tr>
		<tr>
			<td>BMI1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00995536_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCNA2</td>
			<td>Thermofisher scientific</td>
			<td>Hs00996788_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCNB1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01030099_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCNB2</td>
			<td>Thermofisher scientific</td>
			<td>Hs01084593_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCNC</td>
			<td>Thermofisher scientific</td>
			<td>Hs01029304_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCNE1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01026535_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCNF</td>
			<td>Thermofisher scientific</td>
			<td>Hs00171049_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CCR9</td>
			<td>Thermofisher scientific</td>
			<td>Hs01890924_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD10.MME</td>
			<td>Thermofisher scientific</td>
			<td>Hs00153510_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11a</td>
			<td>Thermofisher scientific</td>
			<td>Hs00158218_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c.ITAX</td>
			<td>Thermofisher scientific</td>
			<td>Hs00174217_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD123.IL3RA</td>
			<td>Thermofisher scientific</td>
			<td>Hs00608141_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD133.PROM1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01009250_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD151</td>
			<td>Thermofisher scientific</td>
			<td>Hs00911635_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD220.INSR</td>
			<td>Thermofisher scientific</td>
			<td>Hs00961554_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24.HSA</td>
			<td>Thermofisher scientific</td>
			<td>Hs03044178_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>NCOR1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01094540_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD26.DPP4</td>
			<td>Thermofisher scientific</td>
			<td>Hs00175210_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD274</td>
			<td>Thermofisher scientific</td>
			<td>Hs01125301_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD276</td>
			<td>Thermofisher scientific</td>
			<td>Hs00987207_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD32.FCGR2B</td>
			<td>Thermofisher scientific</td>
			<td>Hs01634996_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD33</td>
			<td>Thermofisher scientific</td>
			<td>Hs01076281_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>Thermofisher scientific</td>
			<td>Hs00990732_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD344.FZD4</td>
			<td>Thermofisher scientific</td>
			<td>Hs00201853_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD352.SLAMF6</td>
			<td>Thermofisher scientific</td>
			<td>Hs01559920_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD38</td>
			<td>Thermofisher scientific</td>
			<td>Hs01120071_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Thermofisher scientific</td>
			<td>Hs01058407_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD41.ITGA2B</td>
			<td>Thermofisher scientific</td>
			<td>Hs01116228_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD49f.ITGA6</td>
			<td>Thermofisher scientific</td>
			<td>Hs01041011_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD56.NCAM1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00941830_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD9</td>
			<td>Thermofisher scientific</td>
			<td>Hs00233521_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD97</td>
			<td>Thermofisher scientific</td>
			<td>Hs00173542_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CD99</td>
			<td>Thermofisher scientific</td>
			<td>Hs00908458_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDK6</td>
			<td>Thermofisher scientific</td>
			<td>Hs01026371_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDKN1A</td>
			<td>Thermofisher scientific</td>
			<td>Hs00355782_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDKN1B</td>
			<td>Thermofisher scientific</td>
			<td>Hs01597588_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CDKN1C</td>
			<td>Thermofisher scientific</td>
			<td>Hs00175938_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CEBPa</td>
			<td>Thermofisher scientific</td>
			<td>Hs00269972_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>CSF1r</td>
			<td>Thermofisher scientific</td>
			<td>Hs00911250_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>CSF2RA</td>
			<td>Thermofisher scientific</td>
			<td>Hs00531296_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>CSF3RA</td>
			<td>Thermofisher scientific</td>
			<td>Hs01114427_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>E2A.TCF3</td>
			<td>Thermofisher scientific</td>
			<td>Hs00413032_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>EBF1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01092694_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>ENG</td>
			<td>Thermofisher scientific</td>
			<td>Hs00923996_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>EPOR</td>
			<td>Thermofisher scientific</td>
			<td>Hs00959427_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>ERG</td>
			<td>Thermofisher scientific</td>
			<td>Hs01554629_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>FLI1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00956711_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>FLT3</td>
			<td>Thermofisher scientific</td>
			<td>Hs00174690_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>FOXO1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01054576_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>Thermofisher scientific</td>
			<td>Hs02758991_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>GATA1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00231112_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GATA2</td>
			<td>Thermofisher scientific</td>
			<td>Hs00231119_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GATA3</td>
			<td>Thermofisher scientific</td>
			<td>Hs00231122_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>GFI1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00382207_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HES1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01118947_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>HLF</td>
			<td>Thermofisher scientific</td>
			<td>Hs00171406_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HMGA2</td>
			<td>Thermofisher scientific</td>
			<td>Hs00171569_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HOXA5</td>
			<td>Thermofisher scientific</td>
			<td>Hs00430330_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HOXB4</td>
			<td>Thermofisher scientific</td>
			<td>Hs00256884_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>ID2</td>
			<td>Thermofisher scientific</td>
			<td>Hs04187239_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF2BP1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00198023_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF2BP2</td>
			<td>Thermofisher scientific</td>
			<td>Hs01118009_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>IKZF1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00172991_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>IL1RAP</td>
			<td>Thermofisher scientific</td>
			<td>Hs00895050_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>IL2RG</td>
			<td>Thermofisher scientific</td>
			<td>Hs00953624_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>IRF8</td>
			<td>Thermofisher scientific</td>
			<td>Hs00175238_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>ITGB7</td>
			<td>Thermofisher scientific</td>
			<td>Hs01565750_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>KIT</td>
			<td>Thermofisher scientific</td>
			<td>Hs00174029_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lin28B</td>
			<td>Thermofisher scientific</td>
			<td>Hs01013729_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>LMO2</td>
			<td>Thermofisher scientific</td>
			<td>Hs00153473_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>LYL1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01089802_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>Meis1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01017441_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>mKi67</td>
			<td>Thermofisher scientific</td>
			<td>Hs01032443_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>MPL</td>
			<td>Thermofisher scientific</td>
			<td>Hs00180489_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>MPO</td>
			<td>Thermofisher scientific</td>
			<td>Hs00924296_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>NFIB</td>
			<td>Thermofisher scientific</td>
			<td>Hs01029175_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Notch1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01062011_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pten</td>
			<td>Thermofisher scientific</td>
			<td>Hs02621230_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>RAG2</td>
			<td>Thermofisher scientific</td>
			<td>Hs01851142_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>RPS18</td>
			<td>Thermofisher scientific</td>
			<td>Hs01375212_g1</td>
			<td></td>
		</tr>
		<tr>
			<td>RUNX1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00231079_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Shisa2</td>
			<td>Thermofisher scientific</td>
			<td>Hs01590823_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Spi1</td>
			<td>Thermofisher scientific</td>
			<td>Hs02786711_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile.IgH</td>
			<td>Thermofisher scientific</td>
			<td>Hs00378435_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>TAL1</td>
			<td>Thermofisher scientific</td>
			<td>Hs01097987_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>THY1</td>
			<td>Thermofisher scientific</td>
			<td>Hs00264235_s1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tim.3.HAVCR2</td>
			<td>Thermofisher scientific</td>
			<td>Hs00958618_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>VWF</td>
			<td>Thermofisher scientific</td>
			<td>Hs00169795_m1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a combinatorial single-cell approach to characterize the molecular and immunophenotypic heterogeneity of human stem and progenitor populations

免疫表型表征和分子分析长期以来被用来描绘异质性和定义不同的细胞种群。流式细胞法本质上是单细胞检测, 但在分子分析之前, 靶细胞通常是预先分离的, 从而失去单细胞分辨率。单细胞基因表达分析提供了一种方法来了解异质细胞种群中单个细胞之间的分子差异。在大容量细胞分析中, 不同细胞类型的过多会导致稀有细胞的信号偏倚和闭塞, 具有生物学重要性。利用流化指数分选与单细胞基因表达分析相结合, 可以在不损失单细胞分辨率的情况下研究种群, 同时捕获具有中间细胞表面标记表达的细胞, 从而使评估连续曲面标记表达式的相关性。在这里, 我们描述了一种方法, 结合单细胞逆转录定量 PCR (rt-pcr) 和流化酶指数排序, 同时表征细胞种群内的分子和免疫表型异质性。
与单细胞 RNA 测序方法不同的是, 使用具有特定靶向扩增的 qPCR, 可以对低丰度的转录物进行稳健的测量, 减少辍学量, 同时不混淆读深度。此外, 通过直接将单细胞分选成裂解缓冲液, 这种方法可以在一步内进行 cDNA 合成和特定靶向扩增, 以及随后的分子特征与细胞表面的相关性标记表达式。所描述的方法是研究造血单细胞, 但也被成功地用于其他细胞类型。
总之, 本文所述的方法允许对预先选择的基因组进行 mRNA 表达的敏感测量, 并有可能开发用于随后的分子不同亚群的未来分离的协议。

所描述的协议是快速、易于执行和高度可靠的。图 1给出了实验设置的概述。整个协议, 从单细胞的排序, 到特定的目标放大, 基因表达测量和初步分析可以在三天内进行。以热图的形式分析结果的一个例子, 它表示从单细胞基因表达分析中使用96引物和96个细胞从慢性髓性白血病 (CML) 患者或96细胞从老年匹配健康的初步分析数据控件如?...

Watch this Scientific Journal Video about A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations at JoVE.com

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

大体积基因表达测量在异构细胞种群中云个体细胞差异。在这里, 我们描述了一个关于如何通过花期活化细胞分类 (immunophenotypically) 的单细胞基因表达分析和指数排序的协议, 以描绘异质性和分子不同细胞种群的特征。

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

这种方法可以回答干细胞和癌症领域的关键问题，如干细胞异质性的外观以及如何分离敏感癌细胞的治疗。本研究的主要优点是，它结合了技术上强大的单细胞基因表达分析与疫苗病，以进一步下游对目标亚细胞的表征。虽然这种方法可以用来研究癌症和干细胞异质性，但它也可以提供基因启动和血统潜力的见解。要开始协议， 在无 RNA/DNA 的长椅上，通过混合 390 微升核酸酶无核酸酶、17 微升 10%NP-40、2.8 微升 10 毫摩尔 dNTP、10 微升 0.1 摩尔 DTT 和 5.3 微升 RNAse 抑制剂，为 96 孔准备足够的解合缓冲液，额外增加 10%。漩涡和旋转下管。接下来，将四微升的解液缓冲液分发到 96 孔 PCR 板的每个孔中，用粘合膜密封板。向下旋转板以收集底部的液体。将盘子放在冰上，直到细胞分拣最多24小时。一旦 FACS 机器正确设置，通过将至少 100 个目标细胞分类到具有 100 微升染色缓冲液的新微离心管中，对目标群体进行重新分析。FACS 通过记录已排序的样本来分析已排序的单元格，并确保它们最终进入排序门。然后通过将 96 井板中井 A1 的落点居中，设置单单元板排序。居中时，在空的96孔板边缘的所有井中对50至106微米的颗粒进行排序，以确保所有孔在每个孔的中心都有一个细胞。从板上取下粘合膜。将林-CD34加CD38减去细胞的单个细胞分拣成96口井中的92个。在 FACS 排序软件中激活索引排序，保存 CD45RA、CD49F 和 CD90 的每个单元的免疫表型配置文件。分别对两个带 10 个和 20 个细胞的孔进行排序，以在 PCR 放大中进行线性控制。通常使用井 H1 和 H2。保持两个井没有任何细胞，因为没有任何模板控制，通常井H3和H4。用透明胶粘膜密封板，并在重力 300 倍时旋转板一分钟。将盘子扣在干冰上。然后将它们储存在零下80摄氏度。通过添加 632.5 微升两次反应混合、101.2 微升 Taq/上标三、151.8 微升底转组合和 0.7 微升在控制RNA中尖峰，进行逆转录和特定目标放大混合。漩涡溶液，并旋转下来收集管底的液体。将混合物放在冰上，直到准备好添加到样品中。接下来，解冻冰上的落酸板。将先前准备的逆转录和特定目标放大混合物的8.75微升添加到92孔中，包括线性和无模板控制。在其余四口井中加入8.75微升无逆转录控制混合物。然后用透明胶膜密封板，然后旋转板，在底部收集液体。根据前置程序在PCR机中运行板，执行逆转录和特定目标放大。通过将三个微升的测定装载试剂移液到 96 井板的每个井中，准备测定装载板。在测定装载板中，将每个底料的三微升添加到单独的孔中。用胶粘膜密封板并旋转下来。旋转板后，将8微升无核酸水移液到96孔板的所有井中，准备稀释板。在稀释板中加入两个微升放大样品。用胶粘膜密封板。通过旋转板 10 秒钟混合。然后旋转板。接下来，通过仔细将 352 微升主混合与 35.2 微升样品加载试剂混合，准备样品加载混合物。通过将 3.3 微升的装载混合物等到 96 井板的每个井中，准备样品装载板。将稀释样品中的 2.7 微升添加到样品装载板的每个井中。用胶粘膜密封板并旋转下来。拿出一个新的96由96微流体芯片。用带帽子的注射器戳进口，以确保它们可以移动。将注射器的全部体积添加到每个阀中，同时将芯片倾斜到 45 度并按下阀门。使用 IFC 控制器为芯片提供上位。从检测装载板的每个孔中用 4.25 微升加载每个检测入口，避免气泡。如果井中出现气泡，请用移液器尖端将其拆下。继续用样品装载板中每个孔的 4.25 微升加载每个样品入口，避免气泡，如果出现气泡，则用移液器尖端将其清除。使用集成流体电路控制器或 IFC 控制器加载芯片。检查芯片看起来看起来均匀，以及所有腔室都已加载。用胶带接触芯片表面的灰尘。最后，在多路复用微流体基因表达平台上运行芯片。成功运行的热图显示样本中均匀分布的表达式，其强信号约为 7 到 25 CTs。这种控制RNA尖峰的放大曲线验证所有孔的清晰表达在10 CT左右，几乎没有变化，验证了所有细胞的扩增前和qPCR反应是否成功。原理成分分析或PCA，使用尺寸减少来可视化细胞组之间的相似性，可以有助于区分分子上不同的细胞群群，在这里识别四个子群。要识别不同分子簇细胞的免疫性，请将索引FCS文件加载到 FlowJo 中。打开脚本编辑器并粘贴索引脚本，按"运行"。每个单元格现在将分一个门。将每个单元格添加到布局编辑器中，并根据文件中可用的 SCXV 分子定义的分组为它们sample_complete_data.xls。FACS图显示了造血干细胞标记CD90和CD45RA的细胞表面标记表达，可用于区分聚类并分离它们进行功能分析。按照这个程序，可以执行其他技术，如新发现的亚细胞的RNA测序或体外和体内实验，以回答其他问题，如什么分子机制导致这种异质性，以及亚细胞在功能上有何不同。这一策略为研究人员不仅研究正常和恶性造血的异质性铺平了道路，还可用于潜在地分离发现的亚体，以进一步研究它们。

Research

JoVE Journal

Genetics

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

增殖和静止人成纤维细胞的全基因组转录衰变率的测定

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...