JQW, SZ, SD and KC are supported by grant from the National Institutes of Health and the Staman Ogilvie Fund&#8212;Memorial Hermann Foundation.

1. EML细胞培养和使用磁性细胞分选系统和Lin-CD34 +和林-CD34-细胞分离荧光激活细胞分选法<ol><li>制备幼仓鼠肾（BHK）细胞培养基中对干细胞因子集合的： <ol><li>培养BHK细胞中，在37℃，5％CO 2下在细胞培养孵育箱含有10％FBS中为25cm 2烧瓶中（ 表1）的DMEM培养基中。 </li><li>当细胞生长至80 - 90％汇合时，用10ml的PBS洗一次细胞。加入5毫升0.25％的胰蛋白酶-EDTA溶液，以单层和孵育的细胞在室温（RT）下1-5分钟，直到细胞分离。 </li><li>吸取溶液，上下轻轻打散细胞团块。加入5 ml完全DMEM至烧瓶中以终止胰蛋白酶的活性。通过离心收集细胞，在200×g离心5分钟，在室温。 </li><li>除去介质和重悬细胞沉淀在10ml新鲜的BHK细胞培养基。 </l我&gt; <li>传送2毫升细胞悬浮液从步骤1.1.4至一个新的75cm 2的烧瓶中，并加入加入48ml新鲜的BHK细胞培养基的烧瓶中。 </li><li>培养的BHK细胞两天，收集培养基。通道通过0.45μm的过滤器中的介质。储存在-20℃下的介质，直到进一步使用。 </li></ol></li><li> EML细胞培养： <ol><li>培养EML细胞（悬浮）在EML的基本培养基中含有的BHK细胞培养基（ 表1），在37℃，5％CO 2的细胞培养孵化器。 </li><li>保持EML的细胞在低细胞密度（0.5-5×10 5个细胞/ ml）与峰密度小于6×10 5个细胞/ ml。分裂细胞每2-3天以1：5的比例。通过EML细胞传代10代后，轻轻地，丢弃文化。 </li></ol></li><li>枯竭谱系阳性细胞： <ol><li>收获在EML的细胞通过离心分离，在200×g离心FOR 5分钟，并用PBS洗一次细胞。收集细胞，离心，在200×g离心5分钟。 </li><li>重悬细胞，用PBS并用血细胞计数器计数细胞。确定抗体浓度在后续细胞分离步骤，根据所述单元的数目（请参阅由细胞分离系统的提供者所提供的说明）。 </li><li>隔离谱系负（Lin-的）使用谱系抗体混合物（生物素标记的单克隆抗体的鸡尾酒细胞CD5，CD45R（B220），CD11b的，抗Gr-1（LY-6G / C），7-4和泰尔-119 ），并根据制造商的说明磁性活化细胞分选系统。 </li></ol></li><li>分离林CD34 +和林-CD34-细胞： <ol><li>降速Lin-的细胞从步骤1.3.3在200×g离心5分钟。重悬细胞沉淀用PBS并用血细胞计数器计数细胞。 </li><li>用FACS缓冲液洗涤细胞两次和颗粒细胞在200 XG5分钟。 </li><li>标签5的1.5​​ ml离心管中分别编号为1，2，3，4，5。重悬的细胞以每10 6个细胞（每管10 6个细胞）加入100μl的FACS缓冲液中。 </li><li>加1微克抗小鼠CD34 FITC抗体对管1和管2和混合管轻轻。 </li><li>孵育所有试管在4℃下1小时在黑暗中。 </li><li>添加0.25 PE标记的抗SCA1抗体微克和20微升的APC标记的世系鸡尾酒抗体对管1中，0.25的PE缀合的抗SCA1抗体对管3微克和20μl的APC标记的世系鸡尾酒抗体来管4。 </li><li>混合所有的管轻轻孵育的细胞中，在4℃下在黑暗中另外的30分钟。 </li><li>添加300μl的FACS缓冲液至细胞和自旋向下的细胞在200×g离心5分钟。 </li><li>用500μl的FACS缓冲液三次洗细胞。 </li><li>重悬细胞沉淀在500μlFACS卜FFER。 </li><li>使用该单元在管2，3，4和5，用于设置补偿。隔离林SCA + CD34 +和林-SCA-CD34-细胞在试管1使用流式细胞仪咏叹调。 </li></ol></li></ol> 2. RNA制备和图书馆建设的高通量测序<ol><li>隔离，质量分析和RNA定量： <ol><li>提取林CD34 +和林-CD34-细胞分别用Trizol按照制造商的协议总RNA。 </li><li>除去受污染的使用DNA脱氧核糖核酸酶I（DNA酶I），按照生产商的方案。可选地，存储所述的RNA在-80℃下以供进一步使用此步骤。 </li><li>评估根据由供应商提供的说明书使用生物分析总RNA的质量。利用RNA样品与RNA完整性（RIN）的啤酒比9。 </li></ol></li><li>图书馆建设与高通量测序： 注：本协议描述的RNA序列利用Illumina平台。为其他测序平台，不同的文库制备方法是必需的。 <ol><li>使用0.1-4微克的高品质的总RNA，每个样品的文库制备。通常为2的总RNA微克可提取10 5 EML的细胞。 </li><li>使用的RNA测序样品制备系统用于RNA纯化和片段化，第一和第二链cDNA合成，末端修复，3&#39;端腺苷化，衔接子连接并进行PCR扩增，按照从提供者的说明，详细的标准程序。 <ol><li>积极选择聚腺苷酸mRNA的使用寡脱氧胸苷酸磁珠和片段的表达。 </li><li>用随机引物来获得的cDNA，随后合成cDNA的第二链，以产生双股cDNA进行反转录。 </li><li>除去3&#39;突出，并填补了5&#39;由DNA聚合酶出挑。腺苷酸3&#39;末端，以防止cDNA片段从结扎到彼此。</li><li>添加多路分度适配器连接到dscDNA的两端。进行PCR对DNA片段的富集。 </li></ol></li><li>测量A260 / A280，得到约文库的使用分光光度计的浓度信息。 </li><li>评估文库质量和测量用生物分析的DNA片段的大小范围。 </li></ol></li></ol> 3.数据分析对于在这部分使用的软件参考，请参阅（ 表2）。 <ol><li>数据文件的处理用于下游分析： <ol><li>转换.bcl（基调用文件）文件中使用CASAVA软件（Ill​​umina公司，1.8.2版本）来.fastq文件。 <ol><li>火起来的&#39;终端&#39;的Linux系统。转到包含从Illumina的HiSeq2000测序仪中的数据文件中的数据文件夹。假设结果文件夹是“NASboy1 / JiaqianLabData / HiSeq_RUN / 2013_07_11 / 130627_SN860_0309_A_2013-166_H0PW9ADXX /&#39;，类型在图S1A的命令，并输入数据的文件夹。 </li><li>在Linux系统上安装CASAVA 1.8.2。假设outputfolder是“未对齐”，使用图S1B命令编写的配置文件转换。使用--fastq簇数0的选项，以确保只有一个.fastq文件是为每个样品创建。所生成的.fastq文件是在名为.gz格式。将它解压缩下游分析（ 图S1B）。 </li><li>已经产生了“不对齐”文件夹后，进入“未对齐”文件夹（ 图S1C）。 </li><li> 图S1D命令开始转换过程。的&#39;-j&#39;参数提供将要使用的CPU的数量。 </li><li>在系统完成转换过程，请在“未对齐”文件夹（ 图S1E）结果的文件夹。 </li><li> 图S1F命令&lt;/ STRONG&gt;解压缩.fastq.gz文件，将根据每个样品夹.fastq文件。 </li></ol></li></ol></li><li>发现新的成绩单和利用Tuxedo的26套房评估的表达水平： <ol><li>地图的配对末端的RNA序列读取到鼠标的参考基因组（UCSC版MM9，从获得的<a href="http://cufflinks.cbcb.umd.edu/igenomes.html" target="_blank">http://cufflinks.cbcb.umd.edu/igenomes.html</a>采用高顶礼帽软件）（版本1.3.3）27，它使用该领结读取映射器（版本0.12.7）28。高顶礼帽提供与“-no-小说，juncs”选项，以提高表达水平的估计精度。 <ol><li>把.fastq文件所在的映射过程将执行一个文件夹中。假设有2 .fastq文件（重命名为Example1.read1，Example1.read2）用于配对末端测序的样品，使用命令， 如图S2中执行的映射（根据系统设定调整参数）。中的“-p”参数提供将要使用的CPU数目。在“-r”和“-mate-STD-dev的”参数可以从库QC索取或从排列的子集推断读取（ 图S2）。 </li></ol></li><li>组装映射读入使用袖扣软件RNA转录（版本1.3.0）29。使用已知的基因注释文件（使用高顶礼帽相同.gtf文件），并通过高顶礼帽产生.bam文件运行袖扣。 <ol><li>后高顶礼帽运行完毕后，在同一个文件夹中，使用图S3A要运行的命令袖扣构建转录和转录估计表达水平。在“mm9_repeatMasker.gtf&#39;和&#39;GenomeSeqMM9”文件夹中的基因组序列文件可以从UCSC基因组浏览器获得。 </li><li>由此产生的genes.expr和transcripts.expr文件包含的基因和转录本（亚型）的表达式的值。复制和粘贴文件内容到一个Excel文件和处理与电子表格应用程序（ 图S3B）。 </li><li> 如图S3C使用命令以确定新的转录所得&#39;transcripts.gtf&#39;文件的引用“mm9_genes.gtf&#39;文件进行比较。 </li><li>所得.tmap文件中包含的比较结果。复制文件内容粘贴到Excel文件和操作与电子表格应用程序。转录物与类代码“u”指可以被视为“新”相比.gtf文件的参考设置（ 图S3D）。 注：对于下游分析的方便，设置FPKM值至0.1，如果值是低于0.1。 注： 步骤3.2.3 - 3.2.6是可选对于那些谁想要提高小说成绩单“表情估计精度。这将需要更长的时间，因为映射和转录施工需要为r未超过一次。 </li></ol></li><li>运行高顶礼帽使用默认参数，然后运行使用图S3E命令袖扣，以生成.gtf文件。 </li><li>比较所得.gtf文件以使用图S3F命令中的参照基因组.gtf文件。 </li><li>解析结果.tmap文件如在步骤3.2.2.4中描述。复制文件内容粘贴到Excel文件和操作与电子表格应用程序。转录物与类代码“u”指可以被视为“新”相比.gtf文件的参考设置。 </li><li>在步骤3.2.5之后，有可被用作参考.gtf文件夹中的一个.combined.gtf文件。高顶礼帽和袖扣的第二次运行可以如在步骤3.2.1和3.2.2中所述，得到的新的转录本的更准确FPKM估计来进行。 </li></ol></li><li>检测differentiall使用DESeq包30岁表达的基因。 <ol><li> DESeq的输入是原始读计数表。为了得到这样一个表，使用分布式与HTSeq Python包可从HTSeq网站下载的htseq计数脚本（ <a href="http://www-huber.embl.de/users/anders/HTSeq/doc/count.html" target="_blank">http://www-huber.embl.de/users/anders/HTSeq/doc/count.html</a> ） 。 <ol><li>确保samtools，Python和htseq数programsare安装在系统中。通过使用图S4A命令获得高顶礼帽输出原始读取计数器值。 </li><li>准备“Raw_Count_Table.txt”使用Excel，“ExperimentDesign.txt”文件。复制并保存为DESeq了R封装（ 图S4B）.txt格式的内容。 </li><li>在系统中安装R程序。在终端，键入“R”，然后按ENTER.A屏幕信息将appearas显示在图S4C。 </li><li>阅读“Raw_Count_Table.txt&#39;，&#39;用图S4D命令ExperimentDesign.txt“成R。 </li><li>加载利用图S4E命令DESeq包。 </li><li>比化条件R（ 图S4F）。 </li><li>使用图S4G命令就归伯爵表运行负二项分布的测试。 </li><li>使用图S4H命令输出显著差异表达基因的.csv文件。 </li></ol></li></ol></li><li>在整个样品查找转录因子&#39;（TFS）FPKM值使用Excel。相交DE基因表和转录因子表。基因属于这两个表中差异表达的转录因子。 <ol><li>去网站<a href="http://www.bioguo.org/AnimalTFDB/download.php" target="_blank">http://www.bioguo.org/AnimalTFDB/download.php</a>和下载的转录因子。那么查找的DE转录因子在Excel中（&lt;<html> STRONG&gt;图S5）。 </li></ol></li><li>产生.bigwig文件UCSC基因组浏览器的可视化。 <ol><li>从网站上下载“bedtools”软件包<a href="https://github.com/arq5x/bedtools2" target="_blank">https://github.com/arq5x/bedtools2</a>并安装该软件系统中的31。从网站下载UCSC工具“bedGraphToBigWig” <a href="http://hgdownload.cse.ucsc.edu/admin/exe/" target="_blank">http://hgdownload.cse.ucsc.edu/admin/exe/</a>并安装该软件在系统中。 </li><li>在包含.bam文件的文件夹，使用图S6A命令来转换通过高顶礼帽到.bed文件生成.bam文件。 </li><li>该.bed文件产生后，使用图S6B命令来生成.bigwig文件。文件&#39;ChromInfo.txt“可以从以下网址获得：ARGET =“_blank”&gt; http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/chromInfo.txt.gz。 </li><li>观察UCSC基因组浏览器自定义的轨道。请参阅网站<a href="http://genome.ucsc.edu/goldenPath/help/customTrack.html" target="_blank">http://genome.ucsc.edu/goldenPath/help/customTrack.html</a>如何显示使用UCSC基因组浏览器自定义的轨道。 </li></ol></li></ol><img alt="图S1" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig1.jpg" /> 图S1：转换.bcl文件使用CASAVA软件.fastq文件。 <img alt="图S2" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig2.jpg" /> 图S2：读取映射使用高顶礼帽到参考基因组。 <img alt="图S3" fo:con帐篷宽度=“5英寸”SRC =“/文件/ ftp_upload / 52104 / 52104supplementalfig3.jpg”/&gt; 图S3：检测新转录和表达水平的估计。 <img alt="图S4" fo:content-width="3.4in" src="/files/ftp_upload/52104/52104supplementalfig4.jpg" /> 图S4：使用DESeq包呼唤差异表达的基因。 <img alt="图S5" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig5.jpg" /> 图S5：差异表达的转录因子的鉴定。 <img alt="图六" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig6.jpg" /> 图六：转换映射结果进行数据可视化。

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The authors have nothing to disclose.

哺乳动物转录是非常复杂的34-38。 RNA测序技术在转录组分析，新转录本的检测和单核苷酸变异的发现等的研究，越来越重要的作用，具有比其它方法进行基因表达分析的许多优点。如引言中所述，它克服了微阵列的杂交工件和可用于鉴定新的转录物从头 。 RNA的测序中的一个限制是比较Sanger测序相对短的读长。然而，随着测序技术的飞速进步，阅读长度不断增加。在本?...

造血干细胞是驻留主要在成人骨髓龛罕见的血细胞。他们负责生产补充血液所需的细胞和免疫系统1。作为一种干细胞，造血干细胞能自​​我更新和分化。阐明了控制造血干细胞的命运决定的机制，无论是对自我更新或分化，将造血干细胞上的操作对血液病的研究和临床应用2提供了宝贵的指导意见。面对的研究者的一个问题是造血干细胞可以维持并在体外扩增到非常有限的程度;他们的后代的绝大多数部分分化培养2。 为了确定控制自我更新和分化的过程中，在全基因组规模的关键调节剂，我们使用了一个鼠原始造血祖细胞系的EML作为模型系统。日是细胞系来自于小鼠骨髓3,4。当具有不同的生长因子供给，EML的细胞能够分化成红细胞，粒细胞，和淋巴细胞在体外 5。重要的是，该细胞系可以传播中大量含有培养基的干细胞因子（SCF）和仍保持其multipotentiality。 EML的细胞可以被分离成自我更新林的SCA + CD34 +亚群和偏微分根据表面标记物CD34和SCA的6林-SCA-CD34-细胞。类似的短期造血干细胞，SCA + CD34 +细胞能够自我更新。当与SCF，林SCA + CD34 +细胞治疗可迅速再生林SCA + CD34 +和林-SCA-CD34-细胞的混合群，并继续增殖6。两个种群在形态相似，并且具有的c-kit mRNA和蛋白6相似的水平。林SCA-CD34-细胞能够在传播媒体的含IL-3，而不是SCF 3。 Unveilin克在EML中的细胞命运决定，将造血过程中提供更好的理解的细胞和分子机制在早期发育过渡的关键调节剂。 为了研究的自我更新林的SCA + CD34 +和偏微分林SCA-CD34 - 细胞间的潜在的分子差异，我们使用RNA测序，以确定差异表达的基因。特别是，我们专注于转录因子，转录因子是决定细胞命运的关键。 RNA测序是最近发展起来的方法，利用新一代测序的能力（NGS）技术来分析和量化的RNA基因组，从7,8转录。简言之，总RNA是多聚A选择并割裂作为初始template.The RNA模板，然后转换成cDNA使用逆转录酶。为了映射全长的RNA转录物，用完整的，未降解的RNA构建cDNA文库，是非常重要的。对于PUR姿势测序，具体接头序列被添加到cDNA的两端。然后，在大多数情况下，cDNA分子通过PCR扩增和测序的高通量方式进行。 测序后，由此产生的读取可以对齐到参考基因组和转录组数据库。的读取数映射到参考基因进行计数，并且这个信息可以被用来估计的基因表达水平。的读取，也可以组装从头无参考基因组，可实现转录的非生物模型9的研究。 RNA的以次技术也已用于检测剪接异构体10-12，新转录13和基因融合14。除了 ​​对编码蛋 ​​白质的基因的检测，RNA测序，也可用于检测新的和分析的非编码RNA，如转录水平长的非编码RNA 15,16，微小RNA 17的siRNA 等 18。由于的t这种方法的他准确性，它已被用于检测的单核苷酸变异19,20。 的RNA测序技术出现之前，微阵列被用来分析基因表达模式的主要方法。预先设计的探针被合成，随后附着于固相表面，以形成微阵列载玻 ​​片21。 mRNA的提取并转化为cDNA。在反转录过程中，荧光标记的核苷酸被掺入到cDNA和该cDNA可以杂交到微阵列载玻片。从一个特定的点收集到的信号的强度依赖于cDNA的上点21结合的特异性探针的量。用RNA测序技术相比，芯片有几个限制。首先，微阵列依赖于基因注释的预先存在的知识，而RNA测序技术能够检测到新的转录物，在相对较高的背景水平，这限制了它的使用，当歌NE表达水平是低的。此外，RNA测序技术有高得多的动态范围检测（8000倍）7，而，由于背景和信号的饱和度，微阵列的准确性是有限的两个高和低表达基因7,22。最后，芯片探针有不同的杂交效率，一个样品23中比较不同转录本的相对表达水平时，这使结果不可靠。虽然RNA测序过微阵列具有许多优点，它的数据分析是复杂的。这是许多研究人员仍然使用RNA测序的微阵列代替的原因之一。各种生物信息学工具所需的RNA测序数据的处理和分析24。 在几种新一代测序（NGS）的平台上，454，Illumina公司，固体和离子洪流是最广泛使用的。 454是第一个商业化NGS平台。在对比的其它测序平台如Illumina公司和扎实，454平台生成更长的读长（平均700个碱基读取）25。较长的读取是transcriptiome的初始特性更好，由于其较高的装配效率25。 454平台的主要缺点是它的每个序列的兆碱基成本高。 Illumina公司和雄厚的平台生成读取增加的数量和长度短。每个序列的兆碱基的成本比454平台低得多。由于大量的短读取的Illumina的和固体的平台，数据分析是更计算密集的。仪器和试剂用于测序的离子洪流平台的价格比较便宜的测序时间短25。但是，错误率和每序列的兆碱基的成本都较高相比，Illumina的和固体的平台。不同的平台都有自己的优点和缺点，需要不同的方法对数据进行分析。解放军TForm的应基于测序的目的和资金的情况进行选择。 在本文中，我们采取的Illumina RNA测序平台为例。我们使用的EML细胞作为模型系统来调查在EML的细胞的自我更新和分化的关键调节剂，并提供RNA测序文库的构建和数据分析表达水平的计算和新的转录检测的详细方法。我们已经证明在我们以前的出版物，在EML的模型系统2的RNA及以下的研究中，加上功能测试（ 例如，shRNA的击倒）提供一种有效的方法，在理解造血细胞分化的早期阶段的分子机制时，并且可以作为一个模型细胞自我更新和分化的一般分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Invitrogen</td>
			<td>15240-062</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD34 FITC</td>
			<td>eBioscience</td>
			<td>11-0341-81</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly-6A/E (Sca-1) PE</td>
			<td>eBioscience</td>
			<td>12-5981-81</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>APC Mouse Lineage Antibody Cocktail</td>
			<td>BD Biosciences</td>
			<td>558074</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>BD FACSAria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>Special offer sysmtem</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 75cm2</td>
			<td>Corning incorporated</td>
			<td>430641</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 25cm2</td>
			<td>Corning incorporated</td>
			<td>430639</td>
			<td>Cell culture</td>
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		<tr>
			<td>Deoxyribonuclease I, Amplification Grade</td>
			<td>Invitrogen</td>
			<td>18068-015</td>
			<td>Library preparation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HI FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16140071</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Invitrogen</td>
			<td>16050-122</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>HyClone</td>
			<td>SH30228.02</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Lineage Cell Depletion Kit, mouse&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>Isolation of lineage negative cells</td>
		</tr>
		<tr>
			<td>NanoVue Plus spectrophotometer&#160;</td>
			<td>GE Healthcare</td>
			<td>28-9569-62</td>
			<td>Quality control</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#8482; Napco&#8482; 8000 Water-Jacketed CO2 Incubators</td>
			<td>Thermo Scientific</td>
			<td>15-497-002&#160;</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>TRIzol&#174; Reagent</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td>RNA exraction</td>
		</tr>
		<tr>
			<td>TruSeq&#8482; RNA Sample Prep Kit v2 -Set B (48rxn)</td>
			<td>Illumina</td>
			<td>RS-122-2002</td>
			<td>Library preparation</td>
		</tr>
		<tr>
			<td>2100 Electrophoresis Bioanalyzer Instrument&#160;</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td>Quality control</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>0.45 &#181;m Syringe Filters</td>
			<td>Nalgene</td>
			<td>190-2545</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
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identification of key factors regulating self-renewal and differentiation in eml hematopoietic precursor cells by rna-sequencing analysis

是造血干细胞（HSCs）用于临床移植治疗以重建病人的造血系统的许多疾病，如白血病和淋巴瘤。阐明控制造血干细胞的自我更新和分化的机制是用于研究和临床用途应用造血干细胞的重要。然而，这是不可能得到大量的造血干细胞，因为它们不能在体外增殖。为了克服这个障碍，我们使用了一个鼠骨髓衍生的细胞系，在EML（红系，髓系和淋巴细胞）细胞系，如用于这项研究的模型系统。 RNA的测序（RNA片段）已被越来越多地用于代替微阵列的基因表达研究。我们在这里报告使用RNA测序技术来调查潜在的关键因素在EML细胞的自我更新和分化的调节的具体方法。在本文提供的方法是分为三个部分。第一杆吨解释了如何培养EML细胞和独立的林CD34 +和林-CD34-细胞。该协议的第二部分提供了详细的程序总RNA制备和随后的图书馆建设高通量测序。最后一部分介绍的方法进行RNA测序数据的分析和解释了如何使用这些数据来确定林-CD34 +和林-CD34-细胞间差异表达的转录因子。最显著差异表达的转录因子被鉴定为潜在的关键调节控制EML的细胞的自我更新和分化的影响。在本文的讨论部分，我们强调这个实验的成功业绩的关键步骤。 综上所述，本文提供了使用RNA测序技术，以确定自我更新和分化中的EML细胞的潜在调节剂的方法。所确定的关键因素进行体外和我的下游功能分析Ñ ​​体内。

为了分析差异表达基因的林CD34 +和林-CD34-细胞EML，我们使用了RNA测序技术。 如图1所示的程序流程。谱系阴性细胞的磁性细胞分选分离后，我们就分开了林SCA + CD34 +，并使用流式细胞仪咏叹调林SCA-CD34-细胞。林富EML的细胞用抗CD34，抗SCA1和谱系鸡尾酒抗体。只有Lin-被门控的SCA1和CD34的表达分析。两个群体（SCA + CD34 +和SCA-CD34- EML的细胞）可以通过FACS分析（ 图2）6被?...

Watch this Scientific Journal Video about Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis at JoVE.com

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

RNA测序和生物信息学分析被用来确定显著和差异表达的转录因子在小鼠EMLcells林-CD34 +和林-CD34-细胞亚群。这些转录因子可能在确定自我更新林的CD34 +和偏微分林CD34-细胞间的开关发挥重要作用。

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

identification-key-factors-regulating-self-renewal-differentiation

Research

JoVE Journal

Biology

12.2K 次观看.  The University of Texas Graduate School of Biomedical Sciences at Houston.  RNA测序和生物信息学分析被用来确定显著和差异表达的转录因子在小鼠EMLcells林-CD34 +和林-CD34-细胞亚群。这些转录因子可能在确定自我更新林的CD34 +和偏微分林CD34-细胞间的开关发挥重要作用。 

视频: Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

SC1（Pluripotin）使用，以支持在没有LIF的自我更新MESC

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Oct4GiP记者试验研究小鼠胚胎干细胞维持和自我更新的基因调节

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired factors are introduced into cold-liquid ECM-mimic gel which, after subcutaneous injection, solidifies to form an environment mimicking the cancer milieu. This matrix permits the penetration of host cells, such as endothelial cells, and therefore, the formation of vasculature.
Herein we propose a new modified matrigel plug assay, which can be exploited to illustrate the angiogenic potential of a pool of factors secreted by cancer cells, as opposed to a specific factor (e.g., bFGF and VEGF) or agent. The plug containing ECM-mimic gel is utilized to introduce the host (i.e., mouse) with a pool of factors secreted to the C.M. of fast-growing tumor-generating glioblastoma cells. We have previously described an extensive comparison of the angiogenic potential of U-87 MG human glioblastoma and its dormant-derived clone, in this system model, showing induced angiogenesis in the U-87 MG parental cells. The C.M. is prepared by filtering collected media from confluent tissue culture plates of either cell line following 48 hr incubation. Hence, it contains only factors secreted by the cells, without the cells themselves. Described here is the combination of two imaging modalities, microbubbles contrast-enhanced ultrasound imaging and intravital fibered-confocal endomicroscopy, for an accurate, real-time characterization of the extent, morphology and functionality of newly-formed blood vessels within the plugs.

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...