这项工作是由NIH授予1SC2GM092789，-01A1，MS新墨西哥州立大学霍华德·休斯医学院校本科研究项目的研究学者。录影被指挥和生产由Amy Lanasa和协调的菲利普·刘易斯博士在新墨西哥州立大学的创意媒体学院。

程序 1。蚊子饲养<ol><li>后蚊子冈比亚按蚊 G3在养虫株为27.5°C，湿度80％，光/暗周期12:12小时。 </li><li>与啤酒酵母的比例为1:1与地面的猫粮喂养幼虫。 </li><li>对小鼠血后第3天出现产蛋饲料成蚊。 </li></ol> 2。蚊子肠道解剖<ol><li>高压灭菌器的清扫工具（幻灯片和钳）。 </li><li>使用抽气收集50个蚊子，把它们放在CO 2流床（终极Flypad）。冲洗顺序在3个培养皿中含有70％的酒精擦拭蚊子体表面的蚊子标本。 </li><li>放置下的立体显微镜载玻片上的检体。删除肠道。 </li><li>收集50个胆为条件宏基因组DNA的提取。收集50个胆量每宏基因组RNA的提取条件。 </li></ol&gt; 3。宏基因组DNA的提取注意：宏基因组DNA中分离，与后述的变形例中使用的Meta-G-诺姆DNA提取试剂盒（震中Biotechnologies公司）。 <ol><li>将50个蚊子胆300μLTE。匀化1分钟使用生物根PRO200匀浆器（PRO科学公司，美国）在转速2000rpm下在冰上。 </li><li>的准备裂解溶菌酶溶液中加入2μl和1μl核糖核酸酶A在细胞悬浮液。混合涡旋和孵化，在37°C 30分钟。 </li><li>加入300微升元裂解液（2X）和1μl蛋白酶K的管。预混液。简言之脉冲离心管，以确保所有的解决方案是在该管的底部，并在65℃孵育15分钟后，冷却至室温，然后放置在冰上3-5分钟。 </li><li>加入350微升的的MPC蛋白质沉淀试剂管和混合剧烈振荡，持续10秒。 </li><li>颗粒吨他碎片通过离心分离10分钟，以12,000 xg在4℃下</li><li>将上清液转移到一个干净的2毫升管中，并弃去沉淀。 </li><li>向上清液中加入570微升异丙醇。混合通过反转管几次。 </li><li>通过以12,000 xg离心10分钟沉淀DNA在4℃下不松脱DNA沉淀的情况下，除去异丙醇。 </li><li>加入500微升的75％乙醇洗涤沉淀。在4℃下以12,000 xg离心5分钟除去乙醇，而不会干扰DNA沉淀。空气干燥的颗粒在室温下2分钟。注意：不要过度干燥DNA沉淀。 </li><li>在50μl的TE缓冲液中重悬DNA沉淀。 </li><li>验证的大小比较（40 kb的100纳克/微升），在试剂盒中提供的的F粘粒控制DNA的分离的DNA，通过凝胶电泳，在1％琼脂糖凝胶电泳。 </li></ol> 4。总RNA提取注意：清洁工作区域RNaseZap，以减少RNase污染。宏基因组RNA的提取使用TriPure隔离试剂（Roche）进行了小修改的蚊子肠道标本。 <ol><li>把蚊子胆50 2毫升试管中用500μlTriPure的隔离试剂。均化1分钟，在转速2000rpm下使用均化器在冰上。静置5分钟，以允许在RT核蛋白复合物的离解。 </li><li>加入100μl氯仿和旋涡，持续12秒，2分钟，静置在RT。 </li><li>离心10,000 xg离心10分钟。管小心地取出，而不会干扰相分离。 </li><li>水相转移到新管中，加入250μl异丙醇，充分混匀，并在-20°C 20分钟。 </li><li>离心机在4℃下以12,000 xg离心10分钟以沉淀RNA。 </li><li>弃上清没有撞出颗粒。 750μL75％乙醇洗涤沉淀。 </li><li>用移液管除去上清液，而不会干扰沉淀。一红外干燥管，持续2分钟。 </li><li>重悬的RNA 30μL无核酸酶的水。 </li><li>治疗的总RNA用DNase在37℃下20分钟，然后灭活DNA酶孵育在75℃下持续15分钟。用乙醇和重悬在30μl无核酸酶的水的RNA沉淀RNA。 </li><li>总RNA，使用的NanoDrop的数量决定的。 </li></ol> 5。核糖体RNA亏损 注：该协议的开发根据上述方法12,13,15。 <ol><li>核糖体RNA基因片段的PCR扩增 此步骤创建特定样品的rRNA池，将被用作体外转录的模板，以产生反义核糖体RNA（arRNA）探针是免费的rRNA基因中的总RNA样品。 <ol><li>设计并合成引物组合rRNA基因片段PCR（ 表1）。 </li><li>在50个运行PCRμl反应体系中使用Taq DNA聚合酶（Qiagen公司制）与50 ng模板DNA，1×PCR缓冲液，1.5MM的Mg 2氯，0.2μM的引物，35个循环的变性在94℃下持续10秒，退火15秒，在40℃下进行细菌23S的扩增子，和50℃下进行其他的扩增子，和延伸在72℃下1分钟（在72℃下5分钟的最后延伸）。 </li><li>查看PCR产物进行1％琼脂糖凝胶电泳。 </li><li>在50μl洗脱缓冲液洗脱使用QIAquick PCR纯化试剂盒纯化PCR产物。量化使用的NanoDrop的浓度。 </li></ol></li><li> 在生物素标记的反义的rRNA的探针（arRNA）的体外转录。注意：在此步骤中，样品从各种rRNA基因的扩增子的特异性探针使用MEGAscript T7试剂盒合成。 <ol><li>通过混合下面的成分（ 表2），设置了一个20μl反应对每个样品的特定rRNA基因的扩增子。孵育过夜，在37°C。 <stroNG&gt;注：通常的反应产率&gt; 50微克的RNA。 </li><li>加入1μlDNA酶I（包括在试剂盒中）的反应，并在37℃下孵育20分钟，以除去DNA模板。 </li><li>加入100μl100％乙醇中反应，离心机在4℃下以12,000 xg离心10分钟以沉淀，合成arRNA。弃去上清，并用750微升75％乙醇洗涤沉淀。在50μl无核酸酶的水重悬RNA探针。 </li><li>量化使用的NanoDrop的RNA浓度。商店探针在-80℃下</li></ol></li><li> rRNA基因减法与生物素arRNA。注意：此步骤采用生物素化的arRNA杂交rRNA基因中的总RNA样品。由链霉亲和素包被的磁珠的rRNA-arRNA的杂交被捕获。程序是基于参考文献13中描述的协议，与细微的修改。 <ol><li>试剂<ol><li>准备0.1N氢氧化钠，20X钠氯碱的ide-柠檬酸钠（SSC）和1×SSC，20％甲酰胺。 </li></ol></li><li>杂交<ol><li>与细菌16S和23S探针（0.75微克），蚊虫18S和28S探针（0.75微克），1微克的总RNA混合，加入1μlRNase抑制剂，2.5微升20X SSC缓冲液，10微升100％甲酰胺中的200微升PCR管，附加组件的无核酸酶的水至50μl。 </li><li>反应管放置在热循环仪和孵育在70℃下5分钟，以打开二级结构和斜坡下降至25℃下，使用5℃的增量，每个℃下1分钟，以允许杂交rRNA和arRNA探头。 </li></ol></li><li>去除rRNA基因<ol><li>成1.7 ml管，将300微升的链霉亲和素包被的磁珠。将管上的磁分离的机架固定的珠子。除去上清液。在300μl的0.1N NaOH和英里悬浮磁珠X井。管放置返回到磁性机架和移液除去上清液。悬浮磁珠在300μl的1×SSC，拌匀，固定的珠子和吸液管除去上清液。重复洗用300μl的1×SSC一个更多的时间。 </li><li> 3等分珠，每100μL，在1.7毫升管。管放置磁性机架上，并通过移液除去上清液。注：取珠用于3轮捕获生物素标记的rRNA的arRNA杂交。 </li><li>加入50μl的1×SSC，用20％甲酰胺到50μl的杂交反应。转移反应到第一胎圈等分管的，拌匀。在RT下孵育10分钟，以允许由链霉抗生物素蛋白磁珠被捕获的生物素化的探针的rRNA杂种。滑动管在孵化混合，每2分钟。 </li><li>管放置磁性机架上，以固定的珠子，将上清转移至所述第二aliqUOT珠，拌匀和孵化如上。进行第三次捕获通过转移到第三管珠上清液。混合和孵化。 </li><li>非rRNA基因转移上清到一个新的管并进行净化，用RNeasy MinElute套件，除去甲酰胺制造商提供的使用手册。 </li><li>请检查的rRNA基因消耗效率的使用Agilent RNA 6000的Pico芯片试剂盒在生物分析仪（ 图4）。 </li></ol></li></ol></li></ol> 注：如有必要，8 mRNA扩增的rRNA耗尽的RNA样品。

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作者宣称，他们没有竞争的金融利益。

一个复杂的微生物群落居住在蚊子的肠道生态系统的14,16,17。 Metatranscriptomic测序（RNA-seq的）上下文相关的功能信息可以透露讯问的整个微生物的转录4,18。从技术上讲，低聚的d（T）介导的富集的原核mRNA的是不适用的，由于缺乏稳定的聚（A）尾的使者。或者，rRNA的耗尽已用于mRNA的富集。在这里，我们开发了消耗rRNA基因rRNA基因探针，从自己在蚊子的肠道微生物群落宏基因组DNA一?...

新一代测序技术已经极大地推进了宏基因组学研究评估的分类组成和遗传功能的微生物组合。 RNA测序（RNA-Seq的）可以绕过文化为基础的方法，，调查微生物metatranscriptomes在不同的情况下2-5。微生物的RNA-seq的一个主要障碍是丰富表达的困难，没有稳定的聚腺苷酸化的原核细胞的mRNA分子。因此，寡d（T）介导的信使富集是不适用的。删除丰富的rRNA基因是一种替代方法来丰富表达。耗竭商业rRNA基因包，如细菌的mRNA Microexpress富集试剂盒（Ambion），RiboMinus转录分离试剂盒（细菌）（Life Technologies公司），和的mRNA-ONLY原核mRNA分离试剂盒（震中）优先降解与核酸外切酶的rRNA基因，已经使用了除去rRNA的6-8。然而，捕获探针Microexpress或RiboMinus用于除去已知的rRNA从典型的革兰氏阳性菌和革兰氏阴性菌（参见制造商的规格）是良好的，但不兼容从未知的微生物与rRNA。因此，去除效率可能较低宏基因组样品8-10。此外，mRNA丰度的保真度是有问题的核酸外切酶处理时，适用11。总体而言，基于减法rRNA基因消耗较少偏见的和更有效的宏基因组的基因富集设置10-13。 蚊子肠道容纳一个充满活力的微生物群落14。我们有兴趣在蚊子的肠道微生物利用RNA-Seq的功能特征。在一个分离的RNA样品由蚊子胆量，蚊子和微生物的RNA存在。在这里，我们描述了修改后的协议，使用社区的具体rRNA探针能够有效地消耗蚊子和微生物的rRNA基因消减杂交技术。所得mRNARNA-Seq的丰富的样本是适当的。在图1中示出的整体的工作流。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material </td>
 </tr>
 <tr>
 <td>Meta-G-Nome 				DNA isolation kit</td>
 <td>Epicentre</td>
 <td>MGN0910</td>
 <td>Metagenomic 				DNA isolation</td>
 </tr>
 <tr>
 <td>TriPure </td>
 <td>Roche</td>
 <td>11667165001</td>
 <td>Mdetagenomic 				RNA isolation</td>
 </tr>
 <tr>
 <td>MEGAscript 				T7 kit</td>
 <td>Ambion</td>
 <td>AM1334</td>
 <td>In 				vitro synthesis of RNA probes</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 <td>RNase 				free working area </td>
 </tr>
 <tr>
 <td>Biotin-16-UTP</td>
 <td>Roche</td>
 <td>11388908910</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Biotin-11-CTP</td>
 <td>Roche</td>
 <td>4739205001</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Streptavidin 				magnetic beads</td>
 <td>NEB</td>
 <td>S1420S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>Magnetic 				separation rack</td>
 <td>NEB</td>
 <td>S1506S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>RNeasy 				mini kit</td>
 <td>QIAGEN</td>
 <td>74104</td>
 <td>Purification 				of subtracted RNA</td>
 </tr>
 <tr>
 <td>RNase-Free 				DNase Set</td>
 <td>QIAGEN</td>
 <td>79254</td>
 <td>Removal 				DNA contamination </td>
 </tr>
 <tr>
 <td>Agilent 				RNA 6000 Pico Kit</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>5067-1513</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bio-Gen 				PRO200 				Homogenizer</td>
 <td>PRO 				Scientific</td>
 <td>01-01200</td>
 <td>Mosquito 				gut tissue homogenization</td>
 </tr>
 <tr>
 <td>NanoDrop 				1000 Spectrophotometer</td>
 <td>Thermo 				Scientific</td>
 <td></td>
 <td>DNA 				&amp; RNA quantitation</td>
 </tr>
 <tr>
 <td>2100 				Bioanalyzer</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>G2940CA</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 </tbody>
</table>

depletion of ribosomal rna for mosquito gut metagenomic rna-seq

蚊子肠道适应动态的微生物群落昆虫的生命周期的不同阶段之间。表征的遗传能力和功能的肠道社区将提供洞察肠道菌群的影响蚊子的生命特质。宏基因组RNA序列分析转录从各种微生物，微生物群落，已经成为一个重要的工具。信使RNA通常仅包括1-3％的总RNA，而rRNA基因构成约90％。它是具有挑战性的丰富信使RNA从宏基因组微生物的RNA样品的原核表达，因为大多数物种缺乏稳定的poly（A）尾巴。这可以防止寡聚d（T）介导的mRNA隔离。在这里，我们描述了一个协议，采用衍生rRNA基因的捕获探针删除rRNA基因从宏基因组总RNA样品的样品。首先，蚊子和社区从宏基因组DNA样品的微生物和大亚基rRNA基因片段的扩增。然后，在交流了nity特定的生物素化的反义核糖体RNA的探针使用T7 RNA聚合酶在体外合成。的生物素化的rRNA基因探针杂交的总RNA。杂交捕获由链霉亲和素包被的磁珠，从总RNA中移除。基于此减法协议有效地消除蚊子和从总RNA样品中的微生物的rRNA。 mRNA的富集样品被进一步处理的RNA扩增和RNA序列。

该协议包括3个部分：（1）准备rRNA基因PCR宏基因组DNA模板（2）消减杂交技术创造特定的样品采集rRNA探针（3）消耗的rRNA总RNA。高品质的宏基因组DNA和RNA分离的整个过程是必不可少的。修改后的Meta-G-的诺姆DNA隔离协议产生高品质的宏基因组DNA从蚊子的胆量， 如图2所示。它可以是具有挑战性的蚊子胆中分离出高品质的总RNA。这可能是由于存在的各种酶，核糖核酸酶，在蚊子的胆量。我?...

Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

核糖体RNA（rRNA）的消耗协议的开发是为了丰富信使RNA（mRNA），RNA-seq中蚊子的肠道metatranscriptome的。由蚊子和其肠道微生物样品的具体rRNA探针，用于去除rRNA基因通过减法，创建了。协议的性能的影响，可能会导致在约90-99％的rRNA基因的去除。

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

depletion-of-ribosomal-rna-for-mosquito-gut-metagenomic-rna-seq

Research

JoVE Journal

Biology

19.5K 次观看.  New Mexico State University. 核糖体RNA（rRNA）的消耗协议的开发是为了丰富信使RNA（mRNA），RNA-seq中蚊子的肠道metatranscriptome的。由蚊子和其肠道微生物样品的具体rRNA探针，用于去除rRNA基因通过减法，创建了。协议的性能的影响，可能会导致在约90-99％的rRNA基因的去除。

视频: Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

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

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

RNA-seq的凝血酶处理和控制人肺微血管内皮细胞的转录组分析

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

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 ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

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;