The authors thank Nicolas Allegre, Maud Peyny and Jean-Philippe Da Ponte for their excellent technical assistance. This work was supported by the Agence Nationale de la Recherche (ANR-JC-CARDIAC-SPE), the INSERM starting grant, ANR ID-CELL-SPE grant, Equipe FRM and the AFM 15845 grant.

1.飞线代<ol><li>产生两个不同的构建体。在第一构建体中，RpL10A的cDNA（核糖体蛋白亚基L10A）被克隆GFP下游入pUASP-PL-GFP-NTER质粒（从32修改后的版本）。使用种系启动子允许转基因系的更多价用法。 注意:通过克隆使用pPTGAL4质粒的调控序列驱动懒散基因的GAL4（TF）的上游的组织特异性表达获取第二构建体。 </li><li>分别注入质粒导入瓦特1118苍蝇和插入构建成由P-元件插入在飞行基因组（按标准程序）33。 </li><li>为了选择转化收回飞线，在两个不同的染色体结构。最终的TRAP线由为了得到一个双转基因稳定线组合这两条线（遗传杂交）创建。 F0:W-; CYO，UAS EGFP :: RpL10A; MKRS / TM6Bx W-; CYO / SCU;没精打采的Gal4 / TM6B。 F1:W-; CYO，UAS EGFP :: RpL10A;没精打采的Gal4 / TM6B。大规模扩大这条线，并收集所需上演胚胎。 </li></ol> 2.胚胎收集<ol><li>准备含有约40克年轻蝇每笼8大圆筒人口笼（约18万蝇/笼）。 </li><li>继续执行所谓的预层次感，让苍蝇清除发育中的胚胎从他们的输卵管。为此，躺在苍蝇1小时于含固化琼脂和葡萄汁的混合物2 11个厘米直径的陪替氏培养皿并覆盖表面的⅓与新制备的酵母膏。经过3交往葡萄果汁板收集类似的新鲜制作的板真正的胚胎。 注:孵育时间将根据发展的时间窗口影响而有所不同。 </li><li>从笼中取出板并离开它们在相同的温度下需要获得正确发育阶段（例如，3小时产卵+ 10小时ADDIT的时间有理孵化给出了从胚胎期10-13小时后产蛋（AEL））。重悬板内容（胚，酵母膏，死蝇）用50毫升的水用刷子。 </li><li>经过一系列的筛（700微米，355微米，112微米）以分离从死苍蝇胚胎和剩余的身体部位，并收集胚胎保持在较小直径筛子，然后在去离子水中简单冲洗的。不要用每收集超过18个板块，因为它会堵塞滤网。 </li><li>在4.5％的漂白粉Dechorionate胚胎在去离子水中2分钟，用去离子水彻底冲洗30-60秒。孵育在PBS 0.01％吐温20，100微克/毫升放线菌酮的胚胎，在室温下搅拌5-10分钟。 </li><li>纤维素吸水张干胚，并将其转移到离心管。称重管和通过浸入液氮中闪冻胚胎。在这个阶段，将干燥的胚胎可存放数个月，在-80℃。 </li></ol><p类="jove_title"&gt; 3。再加以绿色荧光蛋白抗体的磁性珠体的制备<ol><li>彻底重悬蛋白G磁珠在原瓶通过温和搅拌。 </li><li>转移90微升珠不含RNA酶的管和收集它们上的磁体（30-60秒）。 90微升珠粒需要进行免疫沉淀（IP）与1ml裂解物含60-80毫克/毫升蛋白质。尊重比例为防止珠饱和。根据蛋白质的量使用几个管。 </li><li>在磁体洗涤珠粒以1×PBS中0.01％吐温20（500微升），并收集珠丢弃上清液。加30微克的GFP抗体在350微升PBS中0.01％吐温20的至珠孵育缓慢端超过端混合30分钟，在室温。 </li><li>收集在磁体（30-60秒）珠，弃去上清液，并冲洗在500μl的多核糖体提取缓冲液（HEPES 10mM的，氯化钾150mM的，MgCl 2的5mM的，DTT为0.5mM，加入Triton 1％，放线菌酮100微克/毫升，镨otease抑制剂1×，RNA酶抑制剂100 U）。 </li><li>收集在磁铁珠和加入500μl封闭缓冲液（BSA 0.1微克/微升，酵母tRNA 0.1微克/微升，糖原0.1微克/微升在多核糖体提取缓冲液）。 </li><li>孵育在室温30分钟，并用新鲜封闭缓冲液重复此步骤一次。 </li><li>收集的磁珠和冲洗的500微升多聚核糖体提取缓冲液，然后回忆珠立即进行免疫纯化步骤（第6）。 </li></ol> 4.裂解液的制备<ol><li>在开始之前，设立程序上的多方位速度快珠磨机:2×10秒，5000转，15秒暂停。转印干燥胚胎（1.5克）至15ml管中预冷对含有多核糖体提取缓冲液冰，并立即均匀。均质1.5克的胚胎在4毫升的多核糖体提取缓冲液。 </li><li>两种传播胚胎匀浆预冷的2 mL管和研磨安莉芳操作系统通过运行均化程序。转移溶解物到新鲜预冷的微量离心管在冰上，并通过离心在4℃下以2000 克制备postnuclear上清液10分钟。 </li><li>上清转移到新鲜的预冷的微量离心管在冰上，加入10％的Nonidet P-40的1/100样品体积向上清液（最终浓度= 0.1％），并通过反转管轻轻混合。 </li><li>脉冲离心机在minifuge样品收集液体在管的底部，添加300mM的1,2-Diheptanoyl -sn-甘油基-3-磷酸胆碱（DHPC）（终浓度1/9样品体积= 30毫），轻轻混合它通过颠倒试管，并通过孵育期间颠倒几次在冰上孵育混合物5分钟（混合）。 </li><li>制备后线粒体清液离心在4℃下进行10分钟，以20,000 克 。测量蛋白质浓度通过Bradford方法:在裂解物浓度应介于60-80毫克/毫升。 ŤAKE上清液至新鲜新预冷的微量离心管中，并立即进行预吸收步骤（第5部分）。 </li></ol> 5.预吸收<ol><li>彻底缓慢搅拌重悬磁珠并转移30微升珠粒成微量离心管在RT（30微升珠粒/ ml的1胚胎裂解物）。 </li><li>收集珠上的磁铁，吸管上清，重悬在多核糖体提取缓冲液（500微升）的珠。收集珠上的磁铁，加入胚胎裂解物，并孵育1小时，在4℃下在旋转器上。 <ol><li>除去珠，并保持上清冰上的免疫纯化步骤。免疫纯化前，保持100微升上清液（输入）对全球RNA样品与免疫纯化后收集的RNA样品，通过RT-qPCR的，例如比较。 </li></ol></li></ol> 6.免疫纯化<ol><li>加入1毫升（相当于60-80毫克/毫升的蛋白质）的预吸收裂解物至含有90微升耦合到GFP抗体，封闭的珠不含RNA酶的管。孵育样品在4℃下2小时，轻轻端过端混合在试管旋转器中。另外，进行孵化O / N在4℃。 </li><li>培养结束后，收集的磁珠。使用minifuge降速的珠，然后重悬它们在500μl洗涤缓冲液（HEPES 10mM的，氯化钾350MM，MgCl 2的5mM的，诺乃洗涤剂P-40 1％，DTT 0.5mM的，放线菌酮100微克/毫升，RNA酶抑制剂为100U ），并收集他们的磁铁。重复此步骤两次。 </li><li>改变珠粒到一个新的预冷的无RNase管和洗涤两次。收集上的磁铁珠和通过TRIZOL直接加入1ml至珠进行到RNA提取，并按照制造商的说明。 </li></ol> 7. RNA清理与质量评价<ol><li>使用的RNeasy微型试剂盒按照制造商的说明。在完成后，ELUTËRN​​A与（X）微升RNA酶的水。暖2分钟，在60℃，和存储单元冷冻样品在-80℃下。使用生物分析仪检查RNA的质量（按照制造商的说明）。 </li><li>为了测试TRAPed RNA的特异性，根据制造商的说明（上标III）的3纳克纯化的RNA（输入和IP）执行反向转录。使用定量PCR使用基因特异性引物获得的基因。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+Drosophila+with+transposable+element+vectors.">Genetic transformation of Drosophila with transposable element vectors.</a> Science. 218, 348-353 (1982).</li><li>Ingolia, N. 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=Ribosome+profiling+reveals+pervasive+translation+outside+of+annotated+protein-coding+genes.">Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.</a> Cell reports. 8, 1365-1379 (2014).</li></ol>

作者什么都没有透露。

本文介绍一种改性的翻译核糖体亲和纯化协议（称为"陷阱-RC"）致力于在果蝇胚胎罕见的细胞群的研究。信息被设置在用于特定核糖核酸成功分离适于微阵列或RNA-SEQ分析，所需的生物材料，即 ，1）的数量和优化过程为胚胎裂解和多核糖体提取的关键步骤，收率2）珠/抗体对裂解物的比率为最佳免疫沉淀; 3）步骤允许减少的背景，包括洗涤缓冲液组合物，以便运行的TRAP的rc实验以高特异性和灵敏度。 这种协议产生再生的数据使得它容易地应用到任何其他类型的细胞。这种技术使得可以分析差异基因表达在积极-翻译mRNA的水平，从而铺路理解在一个规范表达<html> IFIC细胞类型在一个特定的时间窗口。但是请注意，蛋白丰度将取决于翻译和退化过程的速率。为TRAP方法的主要限制是它不能测量蛋白质含量以精确定量的方式或以检测翻译后修饰。通过测量沿mRNA的核糖体体密度提高量化，并且在这样做时，在一个细胞类型特异性的方式可以使TRAP结合核糖体足迹一个非常强大的工具。在最近的论文34，在人胚胎肾293细胞中进行这种组合。作者跑核酸酶足迹接着RpL10A的使用链亲和素珠诱导型生物素化形式的亲和纯化。这是一个原则证明，有可能在整个有机体而靶向特定的细胞类型，所述的限制是需要的目的生物物质的量进行核糖体足迹。 "&gt;表演TRAP实验在具有全局转录分析并行将使得有可能给新转录和翻译actively-的RNA。这将是深深信息发生在特定的发育上下文或特定细胞群的潜在转录后机制之间跟踪比例 - 用微RNA的例子。 总之，TRAP是一种高效，特异性和灵敏的用于识别的RNA结合到在细胞特异性方式积极-翻译核糖体方法。这种方法可以在各种生物和组织类型的使用。这里所描述的新的TRAP的rc协议稀有细胞群（总细胞数小于1％）和最终材料的足够用于在全基因组水平的后续分析的量进行了优化。 这种方法的一个可能限制是一个强有力的司机的要求，以产生标记的核糖体在足够量的小样ETE与内源的未标记的人在靶细胞类型。在最近的一项研究25，作者估计10-30％的标记与未标记的核糖体的比例。 为了克服这个问题增加UAS-RpL10A-EGFP拷贝数应大大改进这种平衡。备选地，添加到所描述的遗传背景一个UAS-GAL4基因将放大生产GAL4蛋白质的和间接增加RpL10A-EGFP的表达。这应该有利于更好地占用mRNA上标记的核糖体。 注意，这已经有效的方法，可以更有力通过适应互补的方法带来了更多的定量方面或以发现和解开，这对基因表达的控制至关重要的分子机制作出。

了解发育过程中细胞如何获得特殊性能是至关重要的，以欣赏器官的复杂性，以及他们对病理状况的潜在变化。有一个大的研究推动破译的基因调控网络，通过unrevealing全球基因表达谱underly细胞规范和分化，聚合酶II绑定状态，在调控序列和组蛋白翻译后修饰的转录因子占用。 为了评估基因表达在全球范围内芯片和RNA测序方法的使用。微阵列容许检测mRNA的丰度，而核糖核酸-SEQ是更好地面向通知上的不同类型的RNA包括编码和非编码像微RNA，lncRNAs或snRNAs的RNA。然而，这些技术不能区分积极转录，稳态mRNA的，积极-mRNA翻译，和mRNA进入降解过程。测量稳定ST吃了mRNA水平是已知的蛋白质成分1-3的不良估计。在相反，识别积极-mRNA翻译给出蛋白产生的更精确的图像。 然而，转录研究主要被引导在整个生物体水平通过比较增益或的特定因子的功能丧失与野生型条件4-7或解剖组织，使得基因表达谱难以解释。在细胞靶向工具，亲和纯化和灵敏度的改进的最新进展现在允许执行的全基因组分析在活生物体小细胞群体或甚至单个细胞。 在果蝇中，转基因株系大型集合经由GAL4 / UAS系统8-10使不同的组织和细胞亚群的具体时间和空间定向，得到所需的细胞功能的分子机制的更准确的定量。 11。基于蔗糖梯度离心该方法允许多核糖体级分和测定mRNA的的选择翻译的全基因组时与微阵列或深度测序进行分析。多核糖体隔离可与核酸酶消化，以确定对应于在翻译过程12由核糖体保护RNA片段核糖体的脚印。核糖体足迹增加核糖核酸翻译和RNA序列级别分辨率和核糖体定位定量的准确性，使得能够测量翻译的速率。但是，到目前为止还没有被应用到translatomes的小区固有的隔离，其主要原因是在核糖体足迹过程结束时获得的RNA产量的急剧下降。鉴于RNA的大小选择可产生潜在的错误 - 对从也可以以类似的方式保护不同的RNA的RNP ositives，需要进一步的发展以提高核糖体足迹并使其适应小区特有的方法。 这种方法之一，被称为翻译核糖体亲和纯化（TRAP）已在2008年已描述由海曼等人 。并应用于从小鼠的神经元的一个子集隔离translatome。通过以细胞类型特异性的方式表位标记核糖体蛋白质，核糖可以选择性地纯化无细胞解离和排序的费力的工序。在这种情况下，60S核糖体蛋白L10A在核糖体的表面，具有标记的eGFP 13。自2008年以来，一些研究已经用这种方法在不同的物种，从小鼠14-19 为 X. 蟾 20,21，斑马鱼22,23和拟南芥24。该陷阱的方法也已经适应了果蝇模型，并使用普里FY细胞类型特异性从神经元细胞25和星形胶质细胞26的mRNA。多功能二进制GAL4 / UAS系统被用来表示在一个组织特异性方式GFP标记RpL10A。成蝇首脑进行解剖，以执行从（由泛神经ELAV-Gal4的驱动程序有针对性的）神经细胞和孤立的RNA核糖体的选择进行了测序。大量的上调基因对应于那些已知待表达在神经系统，这表明该方法具有良好的特异性和促使我们以使其适应稀有细胞群在显影果蝇胚胎本（小于1细胞的％） 。 在果蝇模型中，胚胎阶段是首选发育过程的研究模型，作为分子的行动者和形态发生运动是进化上保守。在这个模型中迄今开展的唯一的组织特异性转录的方法使用电池或核这样已执行rting并只能够研究稳态转录27-31，创造一个需要专用于组织/细胞特异性translatome分析在果蝇胚胎的方法。在这里，我们报告奉献给果蝇胚胎的第一个陷阱协议。用这种方法，从事合mRNA翻译在每个胚胎周围100肌细胞的一个非常有限的细胞群被成功分离高质量和特异性。二进制GAL4 / UAS系统用于驱动在肌肉的亚群GFP标记RpL10A的表达无任何毒性，没有明显的表型或发育迟缓如前所示25。 果蝇胚胎具有每hemisegment 30的肌肉，这将引起肌肉组织幼虫。通过使用发现的懒散身份基因的附近的调节区，6出来的30多成核的肌肉的有针对性的。在该测定中，核糖体使用的是翻译延伸固定在mRNA上抑制剂放线菌酮。胞质提取物随后用于亲和纯化用GFP抗体 - 包被的磁珠。纯化的RNA质量利用生物分析仪进行了验证。定量逆转录PCR（RT-qPCR的）被用来确定特异性和mRNA分离灵敏度和证明我们的优化的协议是高效率的。

<table><tbody><tr><td>HEPES</td><td>Sigma&amp;nbsp;</td><td>H4034-1006</td><td></td></tr><tr><td>1,2-diheptanoyl-sn-glycero-3-phosphocholine&amp;nbsp;</td><td>Avanti Polar Lipids</td><td>850306P</td><td></td></tr><tr><td>Glycogen</td><td>thermo scientific&amp;nbsp;</td><td>R0561</td><td></td></tr><tr><td>Purified BSA 100X&amp;nbsp;</td><td>Biolabs</td><td>B9001</td><td></td></tr><tr><td>Yeast tRNA&amp;nbsp;</td><td>Sigma</td><td>R5636</td><td></td></tr><tr><td>Super RNase In&amp;nbsp;</td><td>Ambion</td><td>AM2694</td><td></td></tr><tr><td>Antibody GFP clone N86/38</td><td>UC DAVIS/NIH Neuromab Faculty Antibodies Incorporated</td><td>75-132&amp;nbsp;</td><td></td></tr><tr><td>Nonidet P40&amp;nbsp;</td><td>Roche</td><td>11754595001</td><td></td></tr><tr><td>Precellys 24 Lysis homogenisation&amp;nbsp;</td><td>Bertin Technology</td><td></td><td></td></tr><tr><td>Nuclease free water&amp;nbsp;</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Dynabeads ProteinG</td><td>Invitrogen</td><td>10004D</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Applied Biosystem&amp;nbsp;</td><td>AM96406</td><td></td></tr><tr><td>Magnesium Chloride (MgCl2)&amp;nbsp;</td><td>Applied Biosystem&amp;nbsp;</td><td>AM95306</td><td></td></tr><tr><td>Cellulose waddin unbleached&amp;nbsp;</td><td>VWR</td><td>115-2597&amp;nbsp;</td><td></td></tr><tr><td>Sieves 112, 355, 710 um&amp;nbsp;</td><td>Retsch</td><td></td><td></td></tr><tr><td>TRiZol&amp;nbsp;</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>MagRack 6</td><td>GE HealthCare&amp;nbsp;</td><td>28948964</td><td></td></tr><tr><td>Low binding filter tips&amp;nbsp;</td><td>ClearLine</td><td></td><td></td></tr><tr><td>Rnase free tube 1.5 ml&amp;nbsp;</td><td>ClearLine</td><td>390689DD</td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Fischer Bioreagent&amp;nbsp;</td><td>BP337-500</td><td></td></tr><tr><td>Cycloheximide&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>C1985</td><td></td></tr><tr><td>Protease inhibitor tablets, Mini-Complete, EDTA-free&amp;nbsp;</td><td>Roche</td><td>11836170001</td><td></td></tr></tbody></table>

trap-rc, translating ribosome affinity purification from rare cell populations of drosophila embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

果蝇胚胎体细胞肌肉系统由每hemisegment 30的肌肉，并且每个肌肉具有特定的组属性:身份基因，位置，融合事件，附着位点和神经支配多个代码。识别翻译基因在特定的肌肉子会给形成这些特定的肌肉特性所需的蛋白质的信息。从肌肉表达身份基因懒散的亚群使用陷阱基础的方法，纯化RNA（图1A-B）。这种方法的一个主要问题是分离的RNA的质量和特异性。这里报告的优化的协议启用的评估与生物分析仪分析高质量的RNA系统恢复。轮廓获得的节目的RNA没有退化（图1C）。 在周围的TRAP方法开发等的研究，有人提出了数据的特异性。在这里，我们我的mProve抑制背景链接到非特异性核糖核酸至珠或管的结合和通过洗涤缓冲液的优化的方法。为了评估本底水平，并分离懒散表达细胞的效率，我们导致同胚胎阶段的3个重复的RT-qPCR的试验。折叠式富集的4种不同的基因（MEF2，懈怠，普洛斯彼罗，soxNeuro）进行了计算相比输入和归上日RPL32基因（图1D）。这些结果显示泛肌肉基因MEF2和懒散基因的5.6倍富集 2.3倍富集。与此相反，表达在神经系统中的两个基因被耗尽相比输入。非常相似的倍数变化值上观察到3次生物学重复，表明协议是健壮。随着没精打采-Gal4的驱动程序，并开始用1.5克，周围包含100 GFP细胞/胚胎（共150×10 ^ 6阳性1.5×10 ^ 6胚胎获得细胞）。 运行的TRAP实验后，产率为约25-45纳克的视阶段的感兴趣特异性RNA。这种材料是足以运行微阵列分析或使用扩增构建RNA测序文库的RNA序列。效率将用于表达RpL10A-EGFP司机的实力在很大程度上取决于。 <img alt="图1" src="/files/ftp_upload/52985/52985fig1.jpg" /> 图1.质量和从懒散-GAL4&gt; UASRpL10A-EGFP胚胎TRAP隔离RNA特异性评估。 （AB）的阶段-16胚胎表示RpL10A-EGFP表达特别是在每hemisegment（A）中的六个懒散肌肉细胞的共聚焦图像。共定位RpL10A-EGFP的与一般肌肉标记Beta3tubulin上观察到的合并图像 （B）。 （ 三）TRAPed RNA RA的质量控制氮对生物分析仪显示的rRNA 18S和28S完美的完整性。 （ 四）RT-qPCR的分析显示在舞台上，16个胚胎的3生物学重复的TRAP隔离基因实验特异性高。倍数变化的计算方法相比，输入和标准化对RPL32基因存在于所有的肌肉谱系MEF2成绩单是2.3倍富集比输入，而更多的限制没精打采的成绩单是5.6倍富集。神经细胞表达普洛斯彼罗和soxN基因被耗尽的2.2倍和5.5倍，分别为。误差线表示标准偏差（n = 3）。 <a href="https://www.jove.com/files/ftp_upload/52985/52985fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos at JoVE.com

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

TRAP-RC，翻译核糖体亲和纯化的稀有细胞群<em&gt;果蝇</em&gt;胚胎

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

trap-rc-translating-ribosome-affinity-purification-from-rare-cell

Research

JoVE Journal

Biology

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

17.8K 次观看.  University of Clermont-Ferrand. Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos. 

视频: TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

科研

遗传学

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

生物化学

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

中等规模的制备<em&gt;果蝇</em&gt;蛋白质组学实验胚胎提取物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

发育生物学

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, characterizing differences in the translatome (the whole of translated mRNAs) provides an additional layer of information particularly useful when trying to understand the function of RNA regulating or binding proteins. A number of methods for accomplishing this have been developed, including ribosome profiling and polysome analysis. However, both methods carry significant technical challenges and cannot be applied to specific cell populations within a tissue unless combined with additional sorting methods. In contrast, the RiboTag method is a genetic-based, efficient, and technically straightforward alternative that allows the identification of ribosome associated RNAs from specific cell populations without added sorting steps, provided an applicable cell-specific Cre driver is available. This method consists of breeding to generate the genetic models, sample collection, immunoprecipitation, and downstream RNA analyses. Here, we outline this process in adult male mouse germ cells mutant for an RNA binding protein required for male fertility. Special attention is paid to considerations for breeding with a focus on efficient colony management and the generation of correct genetic backgrounds and immunoprecipitation in order to reduce background and optimize output. Discussion of troubleshooting options, additional confirmatory experiments, and potential downstream applications is also included. The presented genetic tools and molecular protocols represent a powerful way to describe the ribosome-associated RNAs of specific cell populations in complex tissues or in systems with aberrant mRNA storage and translation with the goal of informing on the molecular drivers of mutant phenotypes.

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

Quantifying differences in mRNA abundance is a classic approach to understand the impact of a given gene mutation on cell physiology. However, ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

生物学

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

鉴定蛋白质 - 蛋白质相互作用的<em&gt;果蝇</em&gt;成人头由串联亲和纯化（TAP）

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

一个在体内交联方法分离蛋白复合物从果蝇胚胎

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

标记的细胞核从亲和力为基础的隔离<em&gt;果蝇</em&gt;组织的基因表达分析

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

TRAP-RC，翻译核糖体亲和纯化的稀有细胞群

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此视频中的章节

果蝇视觉系统中低丰细胞的纯化

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

中等规模的制备

翻译核糖体亲性纯化(TRAP),用于从体内内皮细胞中分离RNA

翻译核糖体亲和纯化（TRAP）以研究细胞类型特异性尺度下阿拉伯生物基塔利亚纳根发育

雄性小鼠生殖细胞中的核糖核菌免疫沉淀

全山RNA荧光

鉴定蛋白质 - 蛋白质相互作用的

一个在体内交联方法分离蛋白复合物从果蝇胚胎

标记的细胞核从亲和力为基础的隔离