作者要感谢史蒂夫威瑟斯博士和香港明陈提供的DNP - Cellobioside基板。

本议定书开始之前，您将需要您的宏基因组文库在384孔板格式存储。在我们的研究中，我们结合使用噬菌体T1耐TransforMax EPI300 - T1 ř 大肠杆菌 pCC1复制fosmid矢量控制大肠杆菌细胞库的主机和存储我们的板在-80℃11。 1。复制的宏基因组文库板<ol><li>除霜板包含您的图书馆在37℃，约20分钟，或者直到所有的水井都解冻。 </li><li>消毒15分钟的机器人上qPix2使用的紫外灯灭菌功能。 </li><li> 100ug/mL 12.5ug/mL和阿拉伯糖的终浓度在500毫升的试剂瓶，准备与氯霉素LB培养基。每个板块将使用约20ML，再加上作出额外的50毫升，让死体积。 </li><li>设置连接通过媒体瓶无菌管歧管生产商的说明qFill3。适量的媒体计划，并设置它填补了在每孔45uL体积。 </li><li>清除空气中使用清除功能的机器人，直到媒体可见来自歧管的每一个引脚从油管和多方面的。 </li><li>用LB媒体使用qFill3填写所需数量的板块。每盘大约需要20秒，以填补。 </li><li>装入适当的qPix2机器人领域的库板和新鲜的板块。填写相应的试剂清洁浴缸; 2％，在后方浴Micro90，蒸压在中间浴蒸馏水，80％的乙醇在前面浴。 </li><li>使用“复制”计划的qPix2。在软件中，选择适当的头，源盘和目标板的数量和类型。还成立了头之间的复制，通常6个周期，在每次洗澡清洁就足够了。机器容量是在10板，它需要大约15分钟复制一个384针的头（或与一个96针的头部约50分钟）。 注意：有一个选项“爆炒来源”或“爆炒目的地”。建议不使用这些选项的问题与我们的机器人使用他们先前发生。 </li><li>板块一旦被复制，成长在37 ° C，湿度中24小时的板块。因为我们除了与阿拉伯糖诱导的fosmid的高拷贝数，克隆往往增长慢于非诱导克隆。库板返回至-80 ° C 注：在湿度中孵化确保媒体蒸发不会发生在板块的边缘井，所有油井保持一个统一的环境。 </li><li>首先净化水（约50毫升）和80％的乙醇（〜50毫升）的清洁qFill3机器人。拆卸的组件，以及清洁和高压灭菌器在铝箔包裹的瓶子，盖子，和油管。 </li></ol> 2。测量E 的生长大肠杆菌克隆<ol><li>从37 ° C培养箱中取出的板块。删除和杂志加载RapidStak提供的平台上预留的盖子，放置板，最多25板。 注：该板块在堆栈的底部将是第一个被读取。请一定要保持轨道板的顺序，软件不记录。 </li><li>快速Stak取出一本杂志，请确保它是空的，并推到栈板块予以。抓住手柄，都应该加载到杂志的板块。装入RapidStak杂志。 注：该杂志表示板块的A1井应该去哪个角落的角落的一个小螺丝钉。杂志将只安装在一个方向的RapidStak。 </li><li>打开的机器连接到计算机上的SkanIt可再生能源计划。选择“新建会话”，并为其适当命名。选择板型“康宁平底384孔板”。 </li><li>在“板布局”区域，选择“向导”按钮，并选择添加到你的盘子384未知数。 </li><li>在“议定书”的区域，选择“好循环”选项，然后输入384口井。一个“好环”图标，将出现在流树屏幕的左侧。选择图标，并单击“添加”光度测量“。将它设置在600nm处读取。 </li><li>节省您的协议，一个独特的名字和关闭SkanIt稀土程序。 </li><li>在计算机上，打开PolaraRS方案。 </li><li>在主页上，会有表，表中列出的RapidStak设备相连的文书。 VarioSkan应在本表的左侧。 VarioSkan标题下，将有两个环节“RunSession”和“孵化”。点击“RunSession”选项。 </li><li>会出现一个新窗口，检测窗口，在中间的流程图。选择“RunSession”的项目（它应该是唯一的项目目前）。在屏幕右侧的下拉菜单会出现。找到您保存SkanIt在此菜单中运行的可再生能源的名称。 注：此菜单似乎有没有合理的订货。它不是按字母顺序排列，或加入日期，或有关协议在计算机上保存。这意味着你必须寻找才找到自己的所有协议，这是一种痛苦。 </li><li>一旦你选择了您的协议​​，在右上角点击“运行检测”。 </li><li>会弹出一个框，问你，以表示该杂志作为源使用，杂志是空的。在屏幕上下方的方块中，对应于前面的杂志。 [可选：它要求你输入加载板，以确定一个估计的时间，当局将会采取什么。这个估计是通常的方式。] </li><li>确保RapidStak和VarioSkan打开，按“OK”。 </li><li> RapidStak将自动加载到您的板酶标仪，允许连续测量板。要阅读25板，约需50分钟。 注：建议观察VarioSkan到的第一个装版的机器，以确保正确对齐。如果RapidStak不会加载正确的酶标仪，使用“暂停检测”按钮，在右上角的PolaraRS窗口。 </li><li>一旦VarioSkan是阅读完所有的板，取出完整的杂志和杂志上装载平台。抬起平台外的矩形，杂志应该滑落，留下站在一堆在中间板块。更换盖到相应的板块。 </li></ol> 3。除了以每块板的含量组合<ol><li>准备检测混合使用预制裂解混合10倍的股票（10％TRITON X - 100，10MM，100MM RIS EDTA的），50mm的醋酸钾缓冲在pH值5.5。准备一个股票75mg/mL的DNP - cellobioside基板在DMSO，确保基材充分溶解。检测混合添加的DNP - cellobioside原液的终浓度为0.1mg/ml。每个板块将使用约溶液20ml，再加上作出额外的50毫升，让死体积。 注：DNP - Cellobioside是不易溶于水，因此溶解在DMSO增加溶解度。 DMSO在最后的检测解决方案，但没有发现有明显的效果。 </li><li>设置连接通过媒体瓶无菌管歧管生产商的说明qFill3。适量的媒体计划，并设置它填补了在每孔45uL体积。 </li><li>清除空气中使用清除功能的机器人，直到媒体可见来自歧管的每一个引脚从油管和多方面的。 </li><li>检测混合添加到每个板使用qFill3。每盘大约需要20秒，以填补。 </li><li>一旦检测混合添加，板块在37 °湿度中C为12-16小时。 注：在培养板一旦检测混合添加将导致蒸发板的外井，收益较高的吸光度读数比内井。这可以抵消孵化湿度室板。 </li></ol> 4。读吸光度检测克隆<ol><li>从37 ° C培养箱中取出的板块。删除和杂志加载RapidStak提供的平台上预留的盖子，放置板，最多25板。 注：该板块在堆栈的底部将是第一个被读取。请一定要保持轨道板的顺序，软件不记录。 </li><li>快速Stak取出一本杂志，请确保它是空的，并推到栈板块予以。抓住手柄，都应该加载到杂志的板块。装入RapidStak杂志。 注：该杂志表示板块的A1井应该去哪个角落的角落的一个小螺丝钉。杂志将只安装在一个方向的RapidStak。 </li><li>打开的机器连接到计算机上的SkanIt可再生能源计划。选择“新建会话”，并为其适当命名。选择板型“康宁平底384孔板”。 </li><li>在“板布局”区域，选择“向导”按钮，并选择添加到你的盘子384未知数。 </li><li>在“议定书”的区域，选择“好循环”选项，然后输入384口井。一个“好环”图标，将出现在流树屏幕的左侧。选择图标，并单击“添加”光度测量“。将它设置在400nm的阅读。 </li><li>节省您的协议，一个独特的名字和关闭SkanIt稀土程序。 </li><li>在计算机上，打开PolaraRS方案。 </li><li>在主页上，会有表，表中列出的RapidStak设备相连的文书。 VarioSkan应在本表的左侧。 VarioSkan标题下，将有两个环节“RunSession”和“IncubatE“，点击”RunSession“选项。 </li><li>会出现一个新窗口，检测窗口，在中间的流程图。选择“RunSession”的项目（它应该是唯一的项目目前）。一个下拉菜单就会出现在屏幕右侧。找到您保存SkanIt在此菜单中运行的可再生能源的名称。 </li><li>一旦您选择了您的协议​​，在右上角点击“运行检测”。 </li><li>会弹出一个框，问你，以表示该杂志作为源使用，杂志是空的。在屏幕上下方的方块中，对应于前面的杂志。 [可选：它要求你输入加载板，以确定一个估计的时间，当局将会采取什么。这个估计是通常的方式。] </li><li>确保RapidStak和VarioSkan打开，并按下“确定” </li><li> RapidStak将自动加载到您的板酶标仪，允许连续测量板。 注：建议观察VarioSkan到的第一个装版的机器，以确保正确对齐。如果RapidStak不会加载正确的酶标仪，使用“暂停检测”按钮，在右上角的PolaraRS窗口。 </li><li>一旦VarioSkan是阅读完所有的板，取出完整的杂志和杂志上装载平台。抬起平台外的矩形，杂志应该滑落，留下站在一堆在中间板块。该板可根据您的实验室程序处理。 </li><li>要导出的吸光度读数，开放SkanIt RE软件，并选择“打开一个现有的文件”。 </li><li>选择您保存您的会话所在的目录，按下“+”图标展开列表和标题标题下，将标有“容器1”到“集装箱N”的板块数量而定的选项。选择要分析的第一板块。 </li><li>在页面顶部的菜单栏中，选择“数据处理&gt;报告/导出” </li><li>选择出口所需的选项。 注：我通常选择“板布局”和“出口”光度数据。 </li><li>单击“查看报告”和“文件&gt;保存”。选择所需的目录。输出格式是Microsoft Excel电子表格</li></ol> 5。代表性的成果从单一的384板包含一个阳性克隆吸光度读数的一个例子是如图2所示。阳性克隆比那些不表达纤维素酶的吸光度明显增加。检测时间的差异，以及板的位置，或DNP浓度（可筛选出未溶解的DNP出台）可以影响绝对吸光度读数。相对吸光度读数，如在上述板块平均或列平均吸光度的差异，是一个更强大的纤维素确定阳性克隆的方法。 库板的阳性克隆鉴定后，建议复制到一个新盘二次筛选阳性克隆。这消除了井的位置或板块的变化所产生的影响，并允许更多的阳性克隆之间的直接比较。 <img src="/files/ftp_upload/2461/2461fig1.jpg" alt="图1" /> 图1：高通量的检测流程图表为宏基因组文库克隆到大肠杆菌大肠杆菌和保存于-80 ° C。 <img src="/files/ftp_upload/2461/2461fig2.jpg" alt="图2" /> 图2。吸光度读数从一个384孔板包含一个阳性克隆。纤维素酶阳性克隆可以显着增加了负面克隆吸光度确定。

<ol>
	<li>Rubin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+of+cellulosic+biofuels.">Genomics of cellulosic biofuels.</a> Nature. 454, 841-845 (2008).</li><li>Voget, S., Steele, H. L., Streit, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+metagenome+derived+halotolerant+cellulase.">Characterization of a metagenome derived halotolerant cellulase.</a> J. Biotechnol. 126, 26-36 (2006).</li><li>Duan, C. 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在大肠杆菌中表达了一个高通量筛选，从一个大的插入基因组DNA的宏基因组文库的快速检测纤维素活动大肠杆菌是在本议定书。这个方法是在CMC /刚果红在文献中常用的检测改善。这是解决方案为基础，并允许一锅煮化学筛选与定量分析，使读者从板块的吸光度读数最终输出384孔板。在这个过程的每一步自动化允许超过25每小时384板无监督检查。从软件中的数据可以很容易地导出到Mic...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>qPix2</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>&nbsp;</td>
<td>With 384-pin gridding head</td>
</tr>
<tr>
<td>qFill3</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>&nbsp;</td>
<td>With 384-well manifold</td>
</tr>
<tr>
<td>Varioskan</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RapidStak</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>&nbsp;</td>
<td>Connected to Varioskan</td>
</tr>
<tr>
<td>Micro90 Detergent</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>18100-00</td>
<td>Diluted to 2% in water</td>
</tr>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Major Lab Supplier</td>
<td>&nbsp;</td>
<td>Diluted to 80% in water</td>
</tr>
<tr>
<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0378</td>
<td>12.5mg/mL in ethanol</td>
</tr>
<tr>
<td>LB broth, Miller</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP1426-2</td>
<td>25g/L, autoclaved</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>384-well flat bottom plates</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3680</td>
<td>&nbsp;</td>
<tr>
<td>L-(+)-Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3256</td>
<td>100mg/mL in water</td>
</tr>
<tr>
<td>Potassium Acetate</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>P171</td>
<td>50mM in water, autoclaved, adjusted to pH 5.5 with HCl</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP151</td>
<td>&nbsp;</td>
<tr>
<td>Trizma hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T3253</td>
<td>In TE buffer solution, 100mM</td>
</tr>
<tr>
<td>EDTA disodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E5134</td>
<td>In TE buffer solution, 10mM</td>
</tr>
<tr>
<td>2,4-dinitrophenyl cellobioside</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Provided by Dr. Steve Withers, UBC</td>
</tr>
<tr>
<td>Dimethyl Sulfoxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D8418</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a high throughput screen for biomining cellulase activity from metagenomic libraries

纤维素，有机碳在这个星球上最丰富的来源，具有广泛的工业应用日益重视生物燃料生产 1 。修改或降解纤维素的化学方法通常需要强酸和高温。因此，酶的方法已经成为突出的生物转化过程中。虽然已经有点有效识别来自细菌和真菌菌株的积极纤维素酶，微生物在自然界中的绝大多数抵制实验室培养。环境基因组学，又称宏基因组，筛选方法有很大的希望，在弥合在培育新型生物转化酶的搜索差距。宏基因组筛选方法已成功地从环境恢复新颖纤维素酶作为土壤2，水牛瘤胃3和白蚁后肠4使用羧甲基纤维素钠（CMC）的琼脂板用刚果红染料（5 Teather和木材的方法的基础上）染色不同。然而，中央军委的方法是在吞吐量有限，不定量，并表现出低信号信噪比6。其他方法已经7,8报道，但每次使用的琼脂平板为基础的检测，这是为大型插入基因组文库的高通量筛选的不良。在这里，我们提出了一个解决方案，基于纤维素酶的活动，使用显色二硝基苯酚（DNP）cellobioside基板9屏幕。我们的资料库，克隆到fosmid增加检测的灵敏度，通过拷贝数诱导 10 pCC1复制控制。该方法使用一锅煮化学作为测吸光度提供的最终读数384孔板。定量，灵敏，吞吐量高达每天100X 384孔板使用液体处理和附有堆垛系统酶标仪自动读数。

Watch this Scientific Journal Video about A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries at JoVE.com

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

该协议描述了一种在大肠杆菌中表达了宏基因组文库的纤维素活动的高通量筛选。屏幕解决方案的基础和高度自动化，并采用一锅煮化学与最后一个测吸光度读数在384孔板。

一个Biomining从宏基因组库的纤维素酶的活性高通量筛选

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

a-high-throughput-screen-for-biomining-cellulase-activity-from

Research

JoVE Journal

Biology

15.0K 次观看.  University of British Columbia - UBC. 该协议描述了一种在大肠杆菌中表达了宏基因组文库的纤维素活动的高通量筛选。屏幕解决方案的基础和高度自动化，并采用一锅煮化学与最后一个测吸光度读数在384孔板。

视频: A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

单次读取和配对的最终从10微克总RNA的mRNA - Seq的Illumina的图书馆

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;