Questo lavoro è stato sostenuto da NIH sovvenzione 1SC2GM092789-01A1, e MS è stato uno studioso di ricerca di NMSU Howard Hughes Medical Institution di laurea Programmi di Ricerca. Il video è stato diretto e prodotto da Amy Lanasa e coordinato dal Dr. Philip Lewis con il Creative Media Institute NMSU.

Procedura 1. Mosquito allevamento <ol><li> Posteriore zanzara Anopheles gambiae ceppo G3 in un insettario a 27,5 ° C con 80% di umidità e ciclo ore 12:12 del chiaro / scuro. </li><li> Larve si nutrono con cibo per gatti terra con lievito di birra con rapporto 1:1. </li><li> Pasci le zanzare adulte su topi di sangue al giorno 3 post-emergenza per la produzione di uova. </li></ol> 2. Gut Dissection Mosquito <ol><li> Strumenti di dissezione autoclave (diapositive e pinze). </li><li> Colleziona 50 zanzare utilizzando un aspiratore e metterli sul letto di flusso di CO 2 (Ultimate Flypad). Risciacquare zanzara sequenzialmente un campione in tre piastre di Petri contenenti 70% di etanolo per pulire la superficie zanzara corpo. </li><li> Posizionare il campione su un vetrino sotto un stereomicroscopia. Rimuovere l&#39;intestino. </li><li> Colleziona 50 budella per condizione di isolamento del DNA metagenomica. Colleziona 50 budella per condizione per metagenomica isolamento dell&#39;RNA. </li></ol&gt; 3. DNA Isolation metagenomiche Nota: DNA metagenomiche è isolato con il Meta-G-Nome DNA Isolation Kit (Biotecnologie Epicentre) con la modifica descritta di seguito. <ol><li> Mettere 50 budella di zanzara in 300 microlitri TE. Omogeneizzare loro per 1 min con Bio-Gen PRO200 omogeneizzatore (PRO Scientific Inc, USA) a 2.000 giri velocità su ghiaccio. </li><li> Aggiungere 2 microlitri di Ready-Lyse soluzione di lisozima e 1 ml di RNasi A alla sospensione cellulare. Mescolare nel vortex e incubare a 37 ° C per 30 min. </li><li> Aggiungere 300 pl di Meta-Lysis Solution (2X) e 1 ml di proteinasi K al tubo. Miscelare su vortex. Brevemente impulsi centrifugare la provetta per assicurare che tutta la soluzione è nel fondo del tubo, e incubare a 65 ° C per 15 minuti, raffreddare a temperatura ambiente, poi posto in ghiaccio per 3-5 min. </li><li> Aggiungere 350 microlitri di Reagente Precipitation proteine ​​MPC per il tubo e mescolare nel vortex energicamente per 10 sec. </li><li> Pellet tegli detriti mediante centrifugazione per 10 minuti a 12.000 xg a 4 ° C. </li><li> Trasferire il surnatante in un ambiente pulito da 2 ml tubo, e scartare il pellet. </li><li> Aggiungere 570 ml di isopropanolo al supernatante. Miscelare per inversione della provetta diverse. </li><li> Pellet il DNA mediante centrifugazione per 10 minuti a 12.000 xg a 4 ° C. Rimuovere il isopropanolo senza staccare il pellet di DNA. </li><li> Aggiungere 500 ml di etanolo al 75% per lavare il pellet. Centrifugare per 5 minuti a 12.000 xg a 4 ° C. Rimuovere l&#39;etanolo senza disturbare il pellet di DNA. Asciugare il pellet a temperatura ambiente per 2 min. Nota: Non troppo secco il pellet di DNA. </li><li> Risospendere il pellet di DNA in 50 microlitri di tampone TE. </li><li> Convalidare la dimensione del DNA isolato rispetto al DNA di controllo Fosmid (40 kb; 100 ng / pl) fornito nel kit, tramite elettroforesi su gel di agarosio 1% gel. </li></ol> 4. L&#39;RNA totale isolamento Nota: Pulire l&#39;area di lavorocon RNaseZap per minimizzare la contaminazione RNasi. Metagenomiche RNA è stato isolato da campioni di budello di zanzara con il reagente di isolamento TriPure (Roche) con una piccola modifica. <ol><li> Mettere 50 budella di zanzara in un tubo da 2 ml con 500 Reagente isolamento TriPure. Omogeneizzare per 1 min a velocità di 2000 rpm su ghiaccio utilizzando un omogeneizzatore. Lasciare riposare a temperatura ambiente per 5 minuti per consentire la dissociazione dei complessi nucleoproteici. </li><li> Aggiungere 100 microlitri cloroformio e vortex per 12 sec, e lasciate riposare a temperatura ambiente per 2 min. </li><li> Centrifugare a 10.000 xg per 10 min. Estrarre con attenzione il tubo senza disturbare fasi separate. </li><li> Trasferire fase acquosa in una nuova provetta, aggiungere 250 microlitri isopropanolo, mescolare bene, e mettere a -20 ° C per 20 min. </li><li> Centrifugare a 12.000 xg per 10 min a 4 ° C a RNA precipitato. </li><li> Scartare il sopranatante senza staccare pellet. Lavare il pellet con 750 microlitri di etanolo al 75%. </li><li> Pipettare il sopranatante senza disturbare il pellet. Lair asciugare la provetta per 2 min. </li><li> Risospendere l&#39;RNA con 30 microlitri di acqua priva di nucleasi. </li><li> Trattare gli RNA totali con DNasi a 37 ° C per 20 min, quindi inattivare la DNasi incubando a 75 ° C per 15 min. RNA precipitato con etanolo e risospendere l&#39;RNA in 30 microlitri di acqua priva di nucleasi. </li><li> Determinare la quantità di RNA totale usando un NanoDrop. </li></ol> 5. RNA ribosomiale esaurimento Nota: Il protocollo è stato sviluppato sulla base di metodi descritti in precedenza 12,13,15. <ol><li> Amplificazione PCR di frammenti genici RNA ribosomiale Questo passaggio crea campioni specifici per piscine rRNA, che saranno utilizzati come modelli per la trascrizione in vitro per la produzione di anti-senso RNA ribosomiale (arRNA) sonde che sono gratuiti per rRNA nel campione di RNA totale. <ol><li> Progettare e sintetizzare set di primer per rRNA frammento di PCR (Tabella 1). </li><li> Eseguire PCR in 50reazione microlitri utilizzando DNA polimerasi Taq (Qiagen) con 50 ng di DNA template, 1 x tampone PCR, Mg 1.5mm 2 Cl, 0,2 pM primer con 35 cicli di denaturazione a 94 ° C per 10 sec, 15 sec ricottura a 40 ° C per batterica amplicone 23S, e 50 ° C per gli ampliconi altri, e si estendono a 72 ° C per 1 min (estensione finale a 72 ° C per 5 min). </li><li> Visualizza i prodotti di PCR su gel di agarosio all&#39;1%. </li><li> Purificare prodotti PCR utilizzando il kit di purificazione PCR QIAquick con eluizione in 50 microlitri tampone di eluizione. Quantificare la concentrazione utilizzando NanoDrop. </li></ol></li><li> Trascrizione in vitro di biotina-sonde marcate rRNA anti-senso (arRNA). Nota: In questo passaggio, le sonde del campione specifici di varie ampliconi rRNA sono sintetizzati utilizzando il MEGAscript T7 kit. <ol><li> Impostare una reazione di 20 microlitri per ogni amplicone specifico rRNA campione mescolando gli ingredienti indicati (Tabella 2). Incubare per una notte a 37 ° C. <strong&gt; Nota: in genere le rese di reazione&gt; 50 mcg di RNA. </li><li> Aggiungere 1 ml DNasi I (incluso nel kit) per la reazione e incubare a 37 ° C per 20 minuti per rimuovere il DNA stampo. </li><li> Aggiungere 100 pl di etanolo 100% per la reazione, centrifugare a 12.000 xg per 10 min a 4 ° C per precipitare sintetizzato arRNA. Gettare il surnatante e lavare il pellet con 750 microlitri di etanolo al 75%. Risospendere le sonde di RNA in 50 microlitri di acqua priva di nucleasi. </li><li> Quantificare concentrazione di RNA utilizzando un NanoDrop. Sonde Conservare a -80 ° C. </li></ol></li><li> rRNA sottrazione con biotinilato arRNA. Nota: Questa fase utilizza la arRNA biotinilato di ibridare al rRNA nel campione di RNA totale. Gli rRNA-arRNA ibridi vengono catturati da biglie magnetiche rivestite con streptavidina. La procedura si basa sul protocollo descritto nel riferimento 13 con lievi modifiche. <ol><li> Reagenti <ol><li> Preparare NaOH 0,1 N, 20X cloruri di sodioide-citrato (SSC) e 1 X SSC con il 20% formammide. </li></ol></li><li> Ibridazione <ol><li> Mescolare 1 RNA mcg totali con 16S 23S batteriche e sonde (0,75 mg ciascuno), 18S 28S zanzare e sonde (0,75 mg ciascuna), 1 inibitore microlitri RNasi, 2,5 microlitri tampone SSC 20X, 10 microlitri formammide 100% in un tubo da 200 microlitri PCR , aggiungere acqua priva di nucleasi a 50 microlitri. </li><li> Posizionare il tubo di reazione in un termociclatore ed incubare a 70 ° C per 5 minuti per aprire struttura secondaria e rampa di discesa a 25 ° C con 5 ° C con incrementi di 1 min per ogni ° C per consentire l&#39;ibridazione di sonde rRNA e arRNA. </li></ol></li><li> Rimozione di rRNA <ol><li> Trasferire 300 ul perline rivestite con streptavidina in una provetta 1,7 ml. Posizionare il tubo su un rack di separazione magnetica per immobilizzare le perline. Rimuovere il surnatante. Risospendere le sfere in 300 ml di 0,1 N NaOH e mix bene. Posizionare il tubo di ritorno al rack magnetico e pipettare il surnatante. Risospendere le sfere in 300 ml di 1X SSC, mescolare bene, immobilizzare le perline e pipettare il surnatante. Ripetere il lavaggio con 300 ml di 1X SSC ancora una volta. </li><li> Fai 3 aliquote di perline, 100 pl ciascuno, in 1,7 ml provette. Posizionare il tubo sulla griglia magnetica, e rimuovere il surnatante pipettando. Nota: Le aliquote di perline vengono utilizzate per 3 turni di catturare biotinilati rRNA-arRNA ibridi. </li><li> Aggiungere 50 microlitri 1X SSC con il 20% di formammide nella reazione di 50 microlitri di ibridazione. Trasferire la reazione nel primo tubo aliquota tallone, e mescolare bene. Incubare a temperatura ambiente per 10 min per permettere alle sonde biotinilate-rRNA ibridi da acquisire con perline streptavidina. Flick il tubo ogni 2 minuti per mescolare durante l&#39;incubazione. </li><li> Posizionare il tubo sulla griglia magnetica per immobilizzare le perline, trasferire il sopranatante secondo aliquot di perline, mescolare bene e incubare come sopra. Eseguire la cattura terzo trasferendo supernatante nel terzo tubo di perline. Miscelare e incubare. </li><li> Trasferimento non rRNA supernatante in un nuovo tubo e purificazione condotta utilizzando RNeasy kit MinElute per rimuovere formammide seguendo le istruzioni del produttore. </li><li> Verificare l&#39;efficienza deplezione rRNA utilizzando un kit RNA Agilent 6000 Pico chip su un Bioanalyzer (Figura 4). </li></ol></li></ol></li></ol> Nota: I rRNA impoverito campioni di RNA sono soggette a mRNA amplificazione, se necessario 8.

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Gli autori dichiarano di non avere conflitto di interessi finanziari.

Una comunità microbica complessa risiede nel ecosistema intestinale zanzara 14,16,17. Metatranscriptomic sequenziamento (RNA-seq) può rivelare le informazioni di contesto dipendente funzionale interrogando l&#39;intero trascrittoma microbica 4,18. Tecnicamente, oligo-d (T) di arricchimento mediata mRNA procariotici non è applicabile a causa dell&#39;assenza di stabile poly (A) code dei messaggeri. In alternativa, deplezione rRNA è stato utilizzato per l&#39;arricchimento mRNA. Qui, abbiamo svil...

Di prossima generazione di tecnologia di sequenziamento ha notevolmente avanzato studio metagenomica, consentendo di valutare la composizione tassonomica e la funzionalità genetico di un assemblaggio microbica. RNA-Sequencing (RNA-Seq) 1 può ignorare la cultura a base di metodi per indagare metatranscriptomes microbiche in diversi contesti 2-5. Un notevole ostacolo alla microbica RNA-seq è la difficoltà di arricchire mRNA, come le specie di mRNA procariotici non sono stabilmente poliadenilato. Pertanto, oligo d (T) l&#39;arricchimento mediato messenger non è applicabile. La rimozione di rRNA abbondante è un approccio alternativo per arricchire mRNA. Kit commerciali deplezione rRNA, come Microexpress kit batterica Enrichment mRNA (Ambion), RiboMinus Transcriptome Isolation Kit (batteri) (Life Technologies), e mRNA-ONLY Prokaryotic kit di isolamento di mRNA (Epicentre) che degrada preferenzialmente rRNA con una esonucleasi, sono stati utilizzati per la rimozione di rRNA 6-8. Tuttavia, le sonde di cattura in Microexpresso RiboMinus sono buoni per la rimozione di rRNA noto dalle tipiche dei batteri Gram-positivi e Gram-negativi (vedere le specifiche dei costruttori), ma meno compatibile con rRNA da microbi sconosciuti. Conseguentemente, la rimozione può essere meno efficiente per metagenomiche 8-10 campioni. Inoltre, la fedeltà abbondanza mRNA era discutibile quando il trattamento è stato applicato esonucleasi 11. Nel complesso, la sottrazione a base di esaurimento rRNA era meno di parte e più efficace di arricchimento mRNA nelle impostazioni metagenomiche 10-13. L&#39;intestino zanzara ospita una comunità dinamica microbica 14. Siamo interessati a caratterizzare la funzione del microbioma intestinale zanzara utilizzando RNA-Seq. In un campione di RNA isolato da il coraggio di zanzare, sia RNA zanzara e microbiche sono presenti. Qui, descriviamo un protocollo modificato per utilizzare sonde rRNA specifici della comunità di esaurire in modo efficiente rRNA zanzara e microbica mediante ibridazione sottrattiva. L&#39;mRNA risultantecampioni arricchiti sono adatti per RNA-Seq. Il flusso di lavoro complessivo è rappresentato in figura 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;intestino zanzara ospita comunità microbiche dinamiche in diverse fasi del ciclo di vita dell&#39;insetto. Caratterizzazione della capacità genetica e la funzionalità della comunità intestino si forniscono informazioni sugli effetti del microbiota intestinale su tratti della vita di zanzara. Metagenomiche RNA-Seq è diventato uno strumento importante per analizzare trascrittomi da vari microbi presenti in una comunità microbica. RNA messaggero costituito normalmente solo 1-3% di RNA totale, mentre rRNA costituisce circa il 90%. E &#39;difficile per arricchire RNA messaggero da un campione di RNA metagenomica microbica, perché la maggior parte delle specie di mRNA procariote mancano stabili poli (A) code. Questo impedisce oligo d (T) isolamento dell&#39;mRNA mediata. Qui, descriviamo un protocollo che utilizza campione derivato rRNA catturare sonde per rimuovere rRNA da un metagenomica campione di RNA totale. Per iniziare, sia zanzara e microbiche piccoli e grandi frammenti di rRNA subunità sono amplificati da un campione di DNA metagenomica comunità. Poi, la comunitàcomunitari specifici biotinilati sonde antisenso RNA ribosomiale sono sintetizzati in vitro usando T7 RNA polimerasi. Le sonde biotinilate rRNA sono ibridate l&#39;RNA totale. Gli ibridi sono catturati da streptavidina perle rivestite e rimossi dal RNA totale. Questa sottrazione protocollo basato rimuove efficacemente sia zanzara e rRNA microbica dal campione di RNA totale. Il campione di mRNA arricchito ulteriormente trattato per l&#39;amplificazione di RNA e RNA-Seq.

Il protocollo prevede tre sezioni: (1) la preparazione di modelli di DNA metagenomiche per rRNA PCR, (2) la creazione di sonde di cattura specifici rRNA di esempio, (3) esaurimento di rRNA di RNA totale di ibridazione sottrattiva. Isolamento del DNA di alta qualità metagenomica e RNA è essenziale per l&#39;intero processo. La modifica Meta-G-Nome protocollo di isolamento del DNA produce DNA di alta qualità metagenomica di intestini zanzara, come mostrato in Figura 2. Può essere difficile per isolare...

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 RNA ribosomiale (rRNA), il protocollo è stato sviluppato per esaurimento arricchire RNA messaggero (mRNA) per RNA-seq del metatranscriptome intestino zanzara. Sonde rRNA di esempio specifici, che sono stati utilizzati per rimuovere rRNA mediante sottrazione, sono stati creati dalla zanzara e dei suoi microbi intestinali. Prestazioni del protocollo può comportare la rimozione di circa il 90-99% di rRNA.

L&#39;esaurimento delle RNA ribosomiale per metagenomiche Gut Mosquito RNA-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

Research

JoVE Journal

Biology

19.5K Views.  New Mexico State University. Un RNA ribosomiale (rRNA), il protocollo è stato sviluppato per esaurimento arricchire RNA messaggero (mRNA) per RNA-seq del metatranscriptome intestino zanzara. Sonde rRNA di esempio specifici, che sono stati utilizzati per rimuovere rRNA mediante sottrazione, sono stati creati dalla zanzara e dei suoi microbi intestinali. Prestazioni del protocollo può comportare la rimozione di circa il 90-99% di rRNA.

Video: 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.

Su larga scala Schermi di librerie metagenomiche

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

Ricerca

Biologia

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.

Protocollo per la cura dei Mosquito (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.

Generazione Substrato per endonucleasi di CRISPR / Cas Sistemi

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 Analisi trascrittomi in trombina-trattati e di controllo dell&#39;uomo polmonari cellule endoteliali microvascolari

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.

Sequenziamento di prossima generazione del 16S RNA ribosomiale Gene ampliconi

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 Sperimentale e Bioinformatica Protocollo per analisi di RNA-Seq di photoperiodic diapausa nella zanzara tigre,<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.

Fisiologici Registrazioni e RNA sequenziamento delle appendici gustative del Mosquito febbre gialla<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.

Il chitosano / interferenti RNA nanoparticelle mediata silenziamento genico in Disease Vector Mosquito Larve

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: Cellula-tipo-specifico Epigenome profilatura

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;