Ce travail a été soutenu par le NIH subvention 1SC2GM092789-01A1, et MS était un érudit de la recherche NMSU Howard Hughes Programmes Établissement de recherches médicales de premier cycle. La vidéo a été réalisé et produit par Amy Lanasa et coordonné par le Dr Philip Lewis avec le Creative Media Institute à NMSU.

Procédure 1. Élevage Mosquito <ol><li> Arrière moustique Anopheles gambiae G3 souche dans un insectarium à 27,5 ° C avec une humidité de 80% et le cycle h 12:12 de lumière / obscurité. </li><li> Les larves se nourrissent de la nourriture pour chat au sol avec de la levure de bière avec un rapport de 1:1. </li><li> Nourrir les moustiques adultes sur le sang des souris au jour 3 post-levée pour la production d&#39;œufs. </li></ol> 2. Dissection intestin du moustique <ol><li> Outils de dissection autoclave (diapositives et des pinces). </li><li> Recueillir 50 moustiques à l&#39;aide d&#39;un aspirateur et placez-les sur le lit de CO débit 2 (Ultimate Flypad). Rincer une éprouvette moustique séquentiellement dans trois boîtes de Pétri contenant de l&#39;éthanol à 70% pour nettoyer la surface du corps des moustiques. </li><li> Placer l&#39;échantillon sur une lame de verre sous un stéréomicroscope. Retirez le tube digestif. </li><li> Recueillir 50 tripes par condition pour l&#39;isolation d&#39;ADN métagénomique. Recueillir 50 tripes par condition d&#39;isolement d&#39;ARN métagénomique. </li></ol&gt; 3. Isolement de l&#39;ADN métagénomique Remarque: l&#39;ADN métagénomique est isolé en utilisant la méta-G-Nome ADN Isolation Kit (Biotechnologies Epicentre) avec la modification décrite ci-dessous. <ol><li> Place 50, tripes de moustiques dans 300 pi de TE. Les homogénéiser pendant 1 min à l&#39;aide de Bio-Gen PRO200 homogénéisateur (PRO Scientific Inc, États-Unis) à la vitesse de 2000 tours sur la glace. </li><li> Ajouter 2 ul de solution de lysozyme Prêt-Lyse et 1 pl de RNase A à la suspension cellulaire. Mélanger au vortex et incuber à 37 ° C pendant 30 min. </li><li> Ajouter 300 ul de méta-Solution de Lyse (2X) et 1 ul de protéinase K dans le tube. Mélanger au vortex. Brièvement impulsions centrifuger le tube de sorte que l&#39;ensemble de la solution est dans le fond du tube, et incuber à 65 ° C pendant 15 min, refroidir à température ambiante, puis mettre dans la glace pendant 3-5 min. </li><li> Ajouter 350 ul de réactif MPC Protein Precipitation au tube et vortexer vigoureusement pendant 10 secondes. </li><li> Pellet tIl débris par centrifugation pendant 10 min à 12000 xg à 4 ° C. </li><li> Transférer le surnageant dans un endroit propre de 2 ml tube et jeter le culot. </li><li> Ajouter 570 ul d&#39;isopropanol au surnageant. Mélanger à la fois le tube retournant plusieurs. </li><li> Pellet l&#39;ADN par centrifugation pendant 10 min à 12000 xg à 4 ° C. Retirer l&#39;isopropanol sans déloger le culot d&#39;ADN. </li><li> Ajouter 500 ul d&#39;éthanol 75% à laver le culot. Centrifuger pendant 5 min à 12000 xg à 4 ° C. Éliminer l&#39;éthanol sans perturber le culot d&#39;ADN. Air sécher le culot à température ambiante pendant 2 min. Note: Ne pas trop sécher le culot d&#39;ADN. </li><li> Remettre en suspension le culot d&#39;ADN dans 50 pl de tampon TE. </li><li> Valider la taille de l&#39;ADN isolé par rapport à l&#39;ADN de contrôle fosmide (40 ko, 100 ng / ul) fourni dans le kit, par électrophorèse sur gel sur un gel d&#39;agarose 1%. </li></ol> 4. Isolation d&#39;ARN total Remarque: Nettoyer la zone de travailavec RNaseZap pour minimiser la contamination de RNase. Métagénomiques ARN a été isolé à partir d&#39;échantillons intestinaux moustiques en utilisant le réactif d&#39;isolement Tripure (Roche) avec une modification mineure. <ol><li> Mettre 50 tripes de moustiques dans un tube de 2 ml avec 500 ul de réactif Tripure isolement. Homogénéiser pendant 1 min à vitesse 2000 tours sur la glace à l&#39;aide d&#39;un homogénéisateur. Laisser reposer à température ambiante pendant 5 min pour permettre la dissociation des complexes de nucléoprotéines. </li><li> Ajouter 100 ul de chloroforme et homogénéiser au vortex pendant 12 secondes et laisser reposer à température ambiante pendant 2 min. </li><li> Centrifuger à 10000 g pendant 10 min. Sortez soigneusement le tube sans déranger les phases ont été séparées. </li><li> Transférer la phase aqueuse dans un nouveau tube, ajouter 250 ul d&#39;isopropanol, bien mélanger et mettre à -20 ° C pendant 20 min. </li><li> Centrifuger à 12000 g pendant 10 min à 4 ° C à l&#39;ARN précipité. </li><li> Jeter le surnageant sans déloger granulés. Laver le culot avec 750 ul d&#39;éthanol à 75%. </li><li> Introduire à la pipette le surnageant sans déranger le culot. Air sécher le tube pendant 2 min. </li><li> Remettre en suspension l&#39;ARN avec 30 pl d&#39;eau nucléase libre. </li><li> Traiter les ARN totaux avec de la DNase à 37 ° C pendant 20 min, puis inactiver la DNase par incubation à 75 ° C pendant 15 min. ARN précipité avec de l&#39;éthanol et remettre en suspension l&#39;ARN dans 30 pl d&#39;eau nucléase libre. </li><li> Déterminer la quantité d&#39;ARN total en utilisant un NanoDrop. </li></ol> 5. Ribosomal RNA épuisement Remarque: Le protocole a été élaboré sur la base des méthodes décrites précédemment 12,13,15. <ol><li> Amplification par PCR de l&#39;ARN ribosomal fragments de gènes Cette étape crée pools d&#39;échantillons spécifiques ARNr, qui seront utilisés comme modèles pour la transcription in vitro pour produire des anti-sens ARN ribosomal (arRNA) sondes qui sont complémentaires à l&#39;ARNr de l&#39;échantillon d&#39;ARN total. <ol><li> Concevoir et synthétiser des ensembles d&#39;amorces pour l&#39;ARNr fragment de PCR (tableau 1). </li><li> Exécuter PCR dans 50réaction ul utilisant l&#39;ADN polymérase Taq (Qiagen) avec 50 ng de matrice d&#39;ADN, 1 x tampon PCR, 1,5 mM Mg 2 Cl, 0,2 uM amorces avec 35 cycles de dénaturation à 94 ° C pendant 10 sec, 15 sec de recuit à 40 ° C pour bactérienne amplicon 23S et 50 ° C pour les amplicons d&#39;autres, et s&#39;étendant à 72 ° C pendant 1 min (extension finale à 72 ° C pendant 5 minutes). </li><li> Voir les produits de PCR sur gel d&#39;agarose 1%. </li><li> Purifier produits de PCR utilisant le kit de purification de PCR QIAquick avec élution dans 50 ul de tampon d&#39;élution. Quantifier la concentration en utilisant NanoDrop. </li></ol></li><li> La transcription in vitro de la biotine-sondes marquées ARNr anti-sens (arRNA). Remarque: Dans cette étape, les sondes de prélèvement spécifiques des différents amplicons d&#39;ARNr sont synthétisés en utilisant le kit MEGAscript T7. <ol><li> Mettre en place une réaction de 20 ul pour chaque amplicon spécifique ARNr de l&#39;échantillon en mélangeant les ingrédients ci-dessous (Tableau 2). Incuber une nuit à 37 ° C. <strong&gt; Remarque: en général, les rendements de réaction&gt; 50 pg d&#39;ARN. </li><li> Ajouter 1 ul de DNase I (inclus dans le kit) à la réaction et incuber à 37 ° C pendant 20 min pour éliminer la matrice d&#39;ADN. </li><li> Ajouter 100 ul d&#39;éthanol à 100% pour la réaction, centrifugeuse à 12.000 xg pendant 10 min à 4 ° C pour précipiter synthétisé arRNA. Rejeter le surnageant et laver le culot avec 750 ul d&#39;éthanol à 75%. Remettre les sondes d&#39;ARN dans 50 pi d&#39;eau nucléase libre. </li><li> Quantifier la concentration d&#39;ARN en utilisant une NanoDrop. Sondes Conserver à -80 ° C. </li></ol></li><li> ARNr soustraction avec biotinylé arRNA. Remarque: Cette étape utilise le arRNA biotinylé de s&#39;hybrider à l&#39;ARNr de l&#39;échantillon d&#39;ARN total. Les hybrides ARNr arRNA sont capturés par la streptavidine billes magnétiques revêtues. La procédure est basée sur le protocole décrit dans la référence 13 avec des modifications mineures. <ol><li> Réactifs <ol><li> Préparer NaOH 0,1 N, 20X chlore de sodiumide-citrate (SSC) et 1 X SSC avec 20% de formamide. </li></ol></li><li> Hybridation <ol><li> Mélanger 1 ug d&#39;ARN total avec 16S et 23S sondes (0,75 pg chacun), 18S moustiques et les sondes 28S (0,75 mg chacune), 1 inhibiteur de RNase, 2,5 pl 20X tampon SSC, 10 ul de formamide à 100% dans un 200 tube de PCR ul , ajouter eau sans nucléase à 50 pi. </li><li> Placer le tube de réaction dans un thermocycleur et incuber à 70 ° C pendant 5 minutes pour ouvrir la structure secondaire et de descente de la rampe 25 ° C en utilisant 5 ° C par incréments de 1 min chacune ° C pour permettre l&#39;hybridation de sondes d&#39;ARNr et arRNA. </li></ol></li><li> Suppression de l&#39;ARNr <ol><li> Transfert 300 pi billes revêtues de streptavidine dans un tube de 1,7 ml. Placer le tube sur une grille de séparation magnétique pour immobiliser les billes. Retirer le surnageant. Remettre en suspension les billes dans 300 ul de NaOH 0,1 N et mix bien. Placer le tube arrière du rack magnétique et pipeter le surnageant. Remettre en suspension les perles dans 300 pi de SSC 1X, mélangez bien, immobiliser les billes et pipeter le surnageant. Répéter le lavage avec 300 pl de 1X SSC une fois de plus. </li><li> Assurez-3 aliquotes de perles, 100 ul chacun, dans 1,7 ml tubes. Placer le tube sur le support magnétique, et retirer le surnageant par pipetage. Remarque: Les aliquotes de perles sont utilisées pendant 3 rounds de capture biotinylés ARNr arRNA hybrides. </li><li> Ajouter 50 ul 1X SSC avec 20% de formamide dans la réaction de 50 ul hybridation. Transférer la réaction dans le tube aliquote premier talon, et bien mélanger. Incuber à température ambiante pendant 10 minutes pour permettre aux biotinylés sonde ARNr hybrides à être capturés par des billes de streptavidine. Balayez le tube toutes les 2 minutes pour mélanger pendant l&#39;incubation. </li><li> Placer le tube sur le support magnétique pour immobiliser les billes, transférer le surnageant de la seconde aliquot de perles, bien mélanger et incuber comme ci-dessus. Effectuer la capture troisième surnageant par transfert dans le troisième tube de perles. Mélanger et incuber. </li><li> Transfert non-ARNr surnageant dans un nouveau tube et la purification conduite en utilisant RNeasy kit MinElute pour enlever le formamide suivant le manuel du fabricant. </li><li> Contrôler l&#39;efficacité de l&#39;appauvrissement de l&#39;ARNr en utilisant un Agilent RNA 6000 Pico Kit de puce sur une Bioanalyzer (figure 4). </li></ol></li></ol></li></ol> Remarque: Les ARNr appauvri d&#39;échantillons d&#39;ARN sont soumis à l&#39;ARNm d&#39;amplification si nécessaire 8.

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Les auteurs déclarent n&#39;avoir aucun conflit d&#39;intérêts financiers.

Une communauté microbienne complexe réside dans l&#39;écosystème intestin du moustique, 14,16,17. Metatranscriptomic séquençage (ARN-seq) peut révéler des informations de contexte dépend fonctionnelle en interrogeant la totalité du transcriptome 4,18 microbienne. Techniquement, oligo-d (T) d&#39;enrichissement de l&#39;ARNm procaryote médiation n&#39;est pas applicable en raison de l&#39;absence de stabilité poly (A) queues des messagers. Alternativement, l&#39;appauvrissement de l&#39;...

La technologie de séquençage de nouvelle génération a beaucoup progressé étude métagénomique en permettant d&#39;évaluer la composition taxonomique et génétique d&#39;une fonctionnalité associations microbiennes. Séquençage d&#39;ARN (RNA-Seq) 1 peut contourner culture à base de méthodes pour étudier metatranscriptomes microbiennes dans 2-5 contextes différents. Un obstacle majeur à microbien ARN-seq est la difficulté à enrichir l&#39;ARNm, comme les espèces d&#39;ARNm procaryotes ne sont pas stable polyadénylé. Par conséquent, oligo d (T) l&#39;enrichissement messager médiation n&#39;est pas applicable. Suppression de l&#39;ARNr abondante est une approche alternative pour enrichir l&#39;ARNm. Trousses commerciales d&#39;épuisement ARNr, comme Microexpress kit ARNm enrichissement bactérien (Ambion), RiboMinus Isolation Kit transcriptome (bactéries) (Life Technologies), et l&#39;ARNm UNIQUEMENT kit d&#39;isolation d&#39;ARNm procaryote (Epicentre) qui dégrade préférentiellement ARNr avec une exonucléase, ont été utilisés de suppression de l&#39;ARNr 6-8. Cependant, les sondes de capture dans Microexpressou RiboMinus sont bons pour enlever ARNr connu de types de bactéries Gram-positives et Gram-négatives (voir les spécifications du fabricant), mais moins compatible avec les ARNr des microbes inconnus. Par conséquent, la suppression peut être moins efficace pour les 8-10 des échantillons métagénomique. Par ailleurs, la fidélité abondance de l&#39;ARNm était discutable lorsque le traitement a été appliqué exonucléase 11. Dans l&#39;ensemble, la soustraction basée sur l&#39;appauvrissement de l&#39;ARNr est moins biaisé et plus efficace dans l&#39;enrichissement d&#39;ARNm dans les paramètres métagénomiques 10-13. L&#39;intestin du moustique, accueille une communauté microbienne dynamique 14. Nous cherchons à caractériser la fonction du microbiome intestin du moustique en utilisant l&#39;ARN-Seq. Dans un échantillon d&#39;ARN isolés à partir des entrailles de moustiques, les deux ARN moustiques et des microbes sont présents. Ici, nous décrivons un protocole modifié d&#39;utiliser des sondes ARNr communautaires spécifiques pour épuiser les ARNr efficacement les moustiques et microbienne par hybridation soustractive. L&#39;ARNm résultantéchantillons enrichis sont appropriés pour l&#39;ARN-Seq. Le flux global est représenté dans la figure 1.

<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

L&#39;intestin du moustique, accueille les communautés microbiennes dynamiques à travers les différentes étapes du cycle de vie de l&#39;insecte. Caractérisation de la capacité génétique et la fonctionnalité de la communauté intestin permettra de mieux comprendre les effets du microbiote intestinal sur les traits de vie des moustiques. Métagénomique RNA-Seq est devenu un outil important pour analyser les transcriptomes de divers microbes présents dans une communauté microbienne. L&#39;ARN messager comprend généralement que 1-3% de l&#39;ARN total, tandis que ARNr constitue environ 90%. Il est difficile pour enrichir l&#39;ARN messager à partir d&#39;un échantillon d&#39;ARN microbienne métagénomique, car la plupart des espèces d&#39;ARNm procaryotes n&#39;ont pas stables poly (A) des queues. Cela empêche oligo d (T) isolement d&#39;ARNm médiée. Ici, nous décrivons un protocole qui emploie échantillon dérivé ARNr des sondes de capture pour enlever ARNr partir d&#39;un échantillon d&#39;ARN total de métagénomique. Pour commencer, à la fois contre les moustiques et microbiennes petits et grands fragments d&#39;ARNr sous-unités sont amplifiés à partir d&#39;un échantillon d&#39;ADN métagénomique communauté. Ensuite, la communautécommunautaires spécifiques biotinylés antisens ribosomique sondes d&#39;ARN sont synthétisés in vitro en utilisant l&#39;ARN polymérase T7. Les sondes biotinylées ARNr sont hybridées à l&#39;ARN total. Les hybrides sont capturés par des billes revêtues de streptavidine et retiré de l&#39;ARN total. Ce protocole basé sur la soustraction élimine efficacement à la fois contre les moustiques et l&#39;ARNr microbienne de l&#39;échantillon d&#39;ARN total. L&#39;échantillon enrichi en ARNm est ensuite traité pour l&#39;amplification de l&#39;ARN et ARN-Seq.

Le protocole comprend trois sections: (1) la préparation de matrices d&#39;ADN métagénomique pour l&#39;ARNr PCR, (2) la création d&#39;échantillonnage des sondes spécifiques de l&#39;ARNr de capture, (3) l&#39;épuisement de l&#39;ARNr de l&#39;ARN total par hybridation soustractive. Isolation de haute qualité métagénomique ADN et l&#39;ARN est essentielle pour l&#39;ensemble du processus. Le protocole modifié Meta-G-Nome isolement de l&#39;ADN de haute qualité donne ADN métagénomique de viscères de moust...

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

Un protocole d&#39;ARN ribosomal épuisement (ARNr) a été développé afin d&#39;enrichir l&#39;ARN messager (ARNm) pour l&#39;ARN-seq de la metatranscriptome intestin du moustique. Les sondes de prélèvement ARNr spécifiques, qui ont été utilisés pour retirer l&#39;ARNr par soustraction, ont été créés à partir du moustique et ses microbes intestinaux. Performances du protocole peut entraîner la suppression d&#39;environ 90-99% de l&#39;ARNr.

L&#39;appauvrissement de l&#39;ARN ribosomique pour métagénomique Gut Mosquito ARN-seq

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

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JoVE Journal

Biology

19.5K vues.  New Mexico State University. Un protocole d'ARN ribosomal épuisement (ARNr) a été développé afin d'enrichir l'ARN messager (ARNm) pour l'ARN-seq de la metatranscriptome intestin du moustique. Les sondes de prélèvement ARNr spécifiques, qui ont été utilisés pour retirer l'ARNr par soustraction, ont été créés à partir du moustique et ses microbes intestinaux. Performances du protocole peut entraîner la suppression d'environ 90-99% de l'ARNr.

Vidéo: 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.

Grands écrans des banques métagénomiques

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

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Biologie

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.

Protocole pour l&#39;élevage des moustiques (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 ...

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.

Génération substrat pour des endonucléases de CRISPR / Cas Systems

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.

ARN-seq Analyse des transcriptomes de thrombine traitée et de contrôle de l&#39;homme des cellules endothéliales microvasculaires pulmonaires

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.

La nouvelle génération de séquençage de l&#39;ARN ribosomal 16S gènes 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 ...

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.

Un protocole expérimental et de la bioinformatique pour les analyses d&#39;ARN-Seq de la photopériode diapause dans le Asian Tiger Mosquito,<em&gt; Aedes albopictus</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.

Physiologiques Recordings et ARN séquençage des annexes gustatives du Mosquito fièvre jaune<em&gt; Aedes aegypti</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.

Chitosane / ARN interférant nanoparticules Mediated Gene Silencing dans la maladie vectorielle larves de moustiques

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 : Type-spécifiques des cellules épigénome profilage

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;