这项工作是由美国国立卫生研究院资助R21 DE023311（APM），R56 AI111836（APM＆SGF），R01 AI054928（SGF），和R01 DE017088（SGF）的部分资助。

所有的动物程序都在洛杉矶生物医学研究所批准的机构动物护理和使用委员会（协议011000），并根据对动物伦理治疗健康（NIH）的指导方针全国学院进行。将小鼠关在一个AAALAC认证的工厂位于港 - 加州大学洛杉矶分校的研究和教育学院的校园内。有专职兽医谁专门从事实验动物药品监督他们的照顾。笼养和畜牧业是根据指引的美国公共卫生署出版的"指南实验动物的护理和使用"的规定。每一个试图人道地对待老鼠。的小鼠的存活和健康每天监测三次。显然生病，昏睡小鼠从组隔离和安乐死，以减少痛苦。小鼠戊巴比妥过量（210毫克/千克）安乐死的建议，对Euthanas小组IA美国兽医医学协会。 1.准备的组织，试剂与仪器<ol><li>使用雄性BALB / C小鼠权重20-22克所有的研究。接种每个实验组三只小鼠静脉内用1×10 6酵母相C.白色念珠菌细胞。牺牲动物和收获肾脏在特定时间点后感染。将每个肾成螺旋盖管中，管理单元冷冻在液氮中，并储存在-80℃下购买RNA提取6。 注意:动物实验进行如Xu等人详细描述6。 </li><li>从-80℃冰箱取出肾脏之前，请准备以下试剂和仪器: <ol><li>打开一个总RNA提取试剂盒。添加2-巯基乙醇（1％体积/体积）缓冲RLT拌匀（制备2毫升每种样品）。 </li><li>制备苯酚:氯仿:异戊醇25:24:1（需要600微升的各样品）。 </li><li>标签M型均化管（M-管）和冰浴。 </li><li>标签2 ml的螺旋盖管中，并加入约300μl的锆珠在每个管。 </li><li>检查仪器可供选择:组织离解，珠磨器，台式离心机与适配器50ml试管和96孔板，光度计。 </li></ol></li></ol> 2. RNA提取感染组织<ol><li>删除包含从-80°C冰箱肾管和雪藏。添加1.2毫升缓冲液RLT用2-巯基乙醇对每个肾脏。滗肾脏用缓冲液成M型均化管中。 </li><li>均质使用组织离解器中已预先加载设置RNA_02.01肾脏。 </li><li>离心以1000×g离心在台式离心机在室温下1分钟。 </li><li>转移600μl的匀浆含锆珠螺旋盖管中。保存在冰上剩余的匀浆。 </li><li>添加600微升酚:氯仿:异戊醇25:24:1至管子从以前的步骤。 </li><li>在4℃冷室关闭盖子紧密，涡对珠磨器3分钟。 </li><li>离心管在15000×g离心5分钟，4℃冷室中。 </li><li>水相小心转移到一个新的1.5 ml离心管，用70％的乙醇的等体积混合均匀，然后加载到旋转柱（负载两次）。 </li><li>用700微升缓冲RW冲洗旋转柱一次，接着用两次500μl的缓冲液RPE。在干燥的收集管离心一个额外分钟，以除去残留的液体的离心柱。执行所有离心步骤在8000×g的。 </li><li>洗脱的RNA用50μlH 2 O使用分光光度计测量RNA的浓度为OD 260。 </li></ol> 3.基因表达谱使用数字条形码<ol><li>解冻记者代码集（绿帽管）和捕捉代码集（灰帽筒）上冰。 </li><li>添加130微升杂交缓冲液的重新搬运工代码集，颠倒混合和自旋向下。 </li><li>添加20微升混合物到每个12反应管。 </li><li>加10微克总组织的RNA（以体积5微升）到每个管中。通过移液混合。 </li><li>加入5微升捕捉代码集，以每管。翻转管随后又迅速降速混合。 </li><li>孵育反应在65℃下与加热盖O / N（〜18小时）的热循环仪。 </li><li>从-20取出一个样品盒℃，并让它温至室温。 </li><li>除去两个试剂板从4℃离心机在670×g离心在台式离心机2分钟。 </li><li>设置了预备站下面一步一步的指示在屏幕上，选择高灵敏度的选项。 </li><li>从热循环仪中取出的反应，并立即在准备站上加载。选择要运行的高灵敏度程序（3小时）。 </li><li>当准备站计划完成后，取出硒鼓和密封通道用透明胶带。美联社帘布层矿物油到盒的底部（来产生一种NCounter酒店只，后人不需要此步骤），然后加载盒到数字分析仪。 </li><li>建立数字化仪下面一步一步的指示在屏幕上，选择高分辨率的扫描选项。 </li><li>选择高分辨率（600场）选项，运行扫描程序（〜4.5小时）。 </li><li>当扫描程序完成，下载结果（或选择通过电子邮件收到的结果），并导入原始​​数据转换成制造商的软件。软件会自动检查数据和质量提高的标志，如果数据的质量下降超出正常范围。使用内置功能（可选，按照软件的说明）进行技术性调整，然后将数据导出为Excel文件。 </li><li>正常化使用以下方法之一的数据:从代码集的所有基因的总计数;一个或几个内部对照基因;几何平均值的高表达基因（见讨论）。 </li><li>计算平均表达值的每个基因（如果试验在重复或式三份进行），然后计算出的表达水平的不同的实验组之间的比率。 </li><li> （可选）分析和可视化通过在nSolver软件或聚类程序的内置功能，如MultiExperiment浏览器10的分析结果。 </li></ol>

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This protocol is developed in the laboratory of Dr. Aaron P. Mitchell at Carnegie Mellon University. The publication of this manuscript is sponsored by NanoString Technologies.

该协议开发，优化感染使用nanoString NCounter酒店平台的微生物的转录谱。整个过程，从组织收集到的表达数据，需要不到48小时。动手时间约4小时的12个样品。在这个协议中的一个关键的变量是缓冲RLT的加入到组织上的第一步骤的量。太多缓冲器会稀释匀浆，并导致降低的RNA浓度，而过少缓冲器可能导致苯酚氯仿提取步骤后形成粘性凝胶的，离开RNA回收无水相。缓冲RLT为小鼠组织（假设典型大小）的经验确定最佳体积为:肾（1.2毫升），舌（1.0毫升）和肺（2.0毫升）中。对于病原微生物，特别是这些丝状的形式不断壮大，珠跳动一步，有助于破开厚厚的细胞壁充分释放细胞内容物。在洗脱工序中，为了提高最终核糖核酸浓度，在第一轮洗脱液可加回到柱中，洗脱第二次。大约总的组织的RNA的100微克可以从一个RNeasy旋转柱（〜2微克/微升×50微升）进行回收。如果需要较大的RNA量，第二制备可以从剩余的组织匀浆进行。直到RNA浓度进行了测量不处置剩余的匀浆，以防万一。 根据感染模型中，接种量和病原体菌株，病原体RNA的总的组织的RNA的百分比从0％-2％之间，典型地落在0.05％-0.5％的范围内。例如，10微克从 C总组织的RNA的白色念珠菌感染的肾能产生原始计数等于10毫微克纯C的白色念珠菌 RNA的体外培养（10毫微克/ 10微克= 0.1％）。由于病原RNA的总的组织的RNA的百分比在宽范围内变化，它是很难知道pathoge的量Ñ​​RNA的总RNA的给定量。为了避免浪费代码集（250个基因×192反应，每次反应的费用是约$ 200）上与低于检测水平病原体RNA样品，的RNA质量控制步骤可被执行，以确定各样品中的病原体RNA水平。这可以通过Q-RT-PCR或含几看家基因的小的代码集来完成。 用病原体的基因正常化是表达谱前的关键步骤可以在不同样本间进行比较。有三个用于标准化的常用方法。 1.使用总计数从代码集所有基因。 2.用一个或几个"看家"基因。 3.使用几何平均值（ 第 N N个数的乘积的根）的高表达基因。对于一个大的代码集（&gt; 100个基因），含随机选择探针，采用总计数正常化可以是一个很好的选择，因为大量不相关的基因可能信仰的总计数充分体现RNA的输入量。对于一个小代码集（&lt;100基因）含有探针集中在一个特定的处理（例如菌丝生长，考虑到菌丝生长基因往往被共调节的），选择一个或几个看家基因作为对照用于标准化是必不可少的。 TDH3，一个健壮表达代谢基因，曾和控制的许多实验。第三种方法是方法1和2的混合体，重点用于从高表达基因相等的贡献。 虽然NCounter酒店平台不全基因组，它允许表达水平的定量为高达800个基因在单次分析。因此，选择最能提供信息的基因分析是对纹的成功至关重要。两种方法来选择用于探针已被使用。第一种方法是"基础知识"。已出版的表达和功能的数据的信息被编译以筛选基因是潜在感兴趣的当前研究，如对环境的反应基因6-9。这种方法使体内的分析数据，许多现有的数据集，从而提供上下文数据诠释容易比较。第二种方法是"基于探索"。 A类基因在微生物基因组中，如指定转录因子，激酶或细胞壁蛋白的所有基因的被选择为探针。这种方法允许识别基于所述体内分析数据6新颖毒力因子和发现的新调节的关系。

基因表达的动态保存有关细胞如何响应环境变化的重要信息。在一个微生物感染的情况下，基因表达数据可提供有关如何病原体适应于感染环境造成损害到主机临床相关的见解。在过去的二十年里， 无论是在体外和体内基因表达研究已经产生大量的数据，并奠定了基础，了解感染生物学1-3。然而，目前的分析技术，包括芯片和RNAseq，是不是最佳的病原体基因表达的感染过程中剖析。宿主RNA构成从感染的组织样品中分离的总RNA的压倒性部（通常&gt; 99％）。主机RNA有助于对芯片高背景，而占主导地位序列从RNAseq读取。从感染组织病原体细胞分离，在理论上，可以帮助以富集病原体RNA，但它提出了另外人的问题:它需要大量的感染组织;该过程可能是乏味;而且最重要的，所以很难在冗长的过程，以节省天然状态或RNA的完整性。为了产生过程中真正的感染病原基因的表达更全面，更准确的数据，我们着手开发一个协议，允许少数RNA分子的可靠和具有成本效益的量化在混合RNA群。 NanoString NCounter酒店是最近开发的平台使用数字条形码探针对4，5，该平台具有灵敏度水平比得上QRT-PCR的，但不要求酶促扩增，使基因表达的直接复用的测量。该技术并不全基因组，它可以检测在每个测定高达800种不同的基因。因此，关键是要选择信息的基因作为探针进行性能分析。在这里，我们使用基因表达分析的主要嗡嗡声的研究一个病原体，白色念珠菌，在侵入性感染，例如，以证明:1）的实验程序（协议）的技术细节，2）该方法的灵敏度，再现性和动态范围（代表性的结果），和3）重要考虑用于设计和执行基于这个协议（讨论）的实验。该协议可以很容易地适用于各种病原体，并已在许多感染模型6-9得到成功应用。

<table><tbody><tr><td>gentleMACS Dissociator</td><td>Miltenyl Biotec</td><td>130-093-235</td><td></td></tr><tr><td>gentelMACS M tube</td><td>Miltenyl Biotec</td><td>130-093-236</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>QIAGEN</td><td>74104</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M-3148</td><td></td></tr><tr><td>Phenol:CHCl&lt;sub&gt;3&lt;/sub&gt;:IAA</td><td>Sigma</td><td>P-2069</td><td></td></tr><tr><td>Zirconia beads</td><td>Biospec Products</td><td>11079110zx</td><td></td></tr><tr><td>Mini-Beadbeater-16</td><td>Biospec Products</td><td>607</td><td></td></tr><tr><td>nCounter Analysis system</td><td>nanoString Technologies</td><td></td><td></td></tr><tr><td>nCounter Gene Expression Codesets</td><td>nanoString Technologies</td><td></td><td></td></tr><tr><td>nSolver Analysis tool</td><td>nanoString Technologies</td><td></td><td></td></tr></tbody></table>

gene expression profiling of infecting microbes using a digital bar-coding platform

对于大多数哺乳动物病原体，基因表达谱研究已经通过技术困难限于准确地从感染组织量化病原体基因转录。病原RNA构成从被感染的组织样本中分离出的总RNA的一小部分。这两种芯片和RNAseq技术在产生困难可靠的读取为弱表达病原体的基因。突变的病原体菌株具有降低体内扩散构成了更大的挑战。在这里，我们描述了一种在体内基因表达谱协议，这是非常快速的，极其敏感的和高重现性的。我们在我们的血行播散性念珠菌的鼠模型的真菌病原体白色念珠菌的调查过程中开发了这个协议。使用这个协议，我们已经证明动态调节C.时程肾脏感染期间白色念珠菌基因的表达，并发现了意想不到的功能基因表达反应抗真菌药物治疗的体内 。

体内病原体的基因表达分析的一个主要挑战是获得足够的读取病原体RNA。给定病原体转录物中的总RNA，大量的总RNA的比例很低（&gt; 10微克）具有用于每个反应。该平台具有独特的优势，在这个角度:它采用既捕捉探针和报告探针来增加特异性，那么该主机RNA的压倒性量不会引起噪声的显著水平。 如图1（改编自参考文献6），从背景未感染的组织样品的原始计数（红点）均低于10，而来自两个受感染的组织样品（蓝点）的原始计数所产生的所有上述10.表达数据使用此协议具有很强的可重复性。 如图1（改编自参考文献6），原始计数从两个生物学重复（蓝点，48小时感染后肾样本）具有非常粘质三维相关，与R平方值等于0.945。该平台还提供足够的动态范围以包含天然生物的表达水平（图1，原始计数之间10 1和10 6范围）。 使用探针组指定248环境反应的基因，我们能够看出两个阶段病原体的基因表达（ 图2，改编自参考文献6）的:早期的基因表达反应包括基因的RNA水平的接种和12小时之间显著不同在感染后的样品（P &lt;0.05和&gt;中表达2倍的变化），和一个晚期基因表达响应包括那些在48小时后的感染样品（P中的12小时的时间点之间显著不同的基因与RNA水平&lt;0.05和&gt; 2倍的变化表达）。这些结果表明，C。白色念珠菌基因表达过程中侵入infectio动态监管N A哺乳动物宿主的。 <img alt="图1" src="/files/ftp_upload/53460/53460fig1.jpg" /> 图1.原料从感染的和未感染肾组织计数（改编自参考文献6）。探针计算为两个感染（48小时感染后，蓝色数据点）和一个未感染的（红色数据点）肾脏样品呈现为散情节。 <a href="https://www.jove.com/files/ftp_upload/53460/53460fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53460/53460fig2.jpg" /> C的 图2的表达 白色念珠菌 对环境反应基因侵袭性感染期间在小鼠肾脏入侵248 C.（改编自参考文献6）。改变的表达水平白色念珠菌基因在热米呈现AP格式。一式三份生物的平均值示为上调（黄色）和下调的基因的（蓝色），12，24，和感染后相对于48小时为指接种物水平（0小时）。色彩饱和度表示的表达变化在10的范围内，具有完全饱和倍上调或下调。在热图的部分被扩展为说明代表早期上调的基因（上），晚期基因（中），和早期下调基因（底部）。在这些部分，个别样品分别来说明重现性。我们定义的早期表达变化的接种和12小时样品之间显著的差异。我们定义后期表达的变化为12和48小时样品之间显著的差异。意义是指&gt; 2倍和p值&lt;0.05的变化。每个样品的数据进行标准化的RNA水平从控制基因TDH3启动，计算平均值之前。我们的分配条件允许动感中的一些LLY调节基因落入这两个早期和晚期的表达类。 <a href="https://www.jove.com/files/ftp_upload/53460/53460fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform at JoVE.com

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

gene-expression-profiling-infecting-microbes-using-digital-bar-coding

Research

JoVE Journal

Immunology and Infection

8.0K 次观看.  Carnegie Mellon University. We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

视频: Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

免疫与感染

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

多重条形码图像分析，用于石蜡组织样品单细胞分析中的免疫分析和空间映射表征

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

癌症研究

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

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

发育生物学

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

生物工程

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

生物学

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease.&#160;To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols.&#160;The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future&#160;in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections.

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

用于研究宿主-病原体相互作用的高通量转录组分析

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...