Riconosciamo sostegno da parte Scienze Chimiche, Geoscienze e Biosciences Division, Office of Basic Sciences Energy, Office of Science, US Department of Energy (numero Premio DE-FG02-91ER20021) e National Science Foundation (MCB 0.948.584) (FB). Siamo grati a Bird sig.ra Karen per l&#39;editing del manoscritto.

1. EMS Trattamento Semi di Arabidopsis thaliana sono mutagenizzato utilizzando come agente mutageno metano solfonato di etile (EMS) 3,4, che induce nelle C-to-T genoma cambi derivanti in C / G a T / A mutazioni 5-7. <ol><li> Pesare 0,8 g di semi di Arabidopsis (circa 40.000 semi) che trasportano il marcatore fluorescente organello (specificamente, in questo studio ssGFPHDEL (sequenza segnale-GFP-HDEL tetrapeptide) è stato usato come marcatore ER). </li><li> Trasferire i semi in un tubo da 50 ml Falcon, poi aggiungere 25 ml di acqua distillata. </li><li> Aggiungere 0,2% (v / v) metano solfonato etile. </li><li> Incubare per 16 ore su oscillante miscelatore a bassa velocità. </li><li> Aspirare ed eliminare il liquido in un pallone contenente 1,0 M NaOH per inattivare l&#39;EMS. </li><li> Aggiungere 25 ml di acqua nella provetta Falcon che contiene i semi, chiudere e capovolgere 5 volte per lavare i semi; attendere che tutti i semi si sono stabiliti, quindi aspirare l&#39;acqua e gettare nel 1,0 M Na<html> OH pallone. </li><li> Ripetere la fase di lavaggio fino a 10 volte. </li><li> Dopo l&#39;ultimo lavaggio, risospendere i semi in una quantità minima di acqua. </li><li> Procedere alla sterilizzazione delle sementi aggiungendo 25 ml di candeggina al 10%, si agita energicamente per 30 s. Lasciate che i semi depositerà sul fondo, quindi versare off candeggina e sciacquare con 25 ml di acqua sterile. Versare l&#39;acqua sterile e aggiungere 25 ml di etanolo al 70%. Agitare provette per 30 s. Lasciate che i semi depositerà sul fondo. Versare etanolo e sciacquare con 25 ml di acqua sterile. Ripetete due volte il lavaggio con acqua sterile, poi versate i semi su un piatto di plastica Petri contenente 3 mm Carta filtro e lasciare asciugare sotto il cofano. Conservare a 4 ° C per 2 giorni. </li><li> Piastra i semi sulla ½ MS phytagel (concentrazione media la metà di Murashige e Skoog), 150 mm Petri (circa 250-300 semi per piastra). </li><li> Crescere semi M1 sul piatto per 2 settimane, poi trapiantare nel terreno. </li><li> Raccogliere i semi M2 da singoli impianti per generare linee M1 M2 (1000 linee indipendenti). </li></ol>ove_title &quot;&gt; 2. Screening confocale delle popolazioni M2 e M3 In questa sezione descrive l&#39;osservazione di piantine con un microscopio confocale a fluorescenza o come descritto in precedenza 8. <ol><li> Sessanta semi di ogni linea M2 sono cresciuti da 7 a 10 giorni in plastica piastre di Petri, ½ MS phytagel, sulla stessa piastra vengono coltivate anche 5 piantine di EMS-non trattati (controllo). </li><li> Da cinque a dieci cotiledoni sono montati con il lato abassiale verso l&#39;obiettivo (40X) su un vetrino da microscopio e chiuso con coprioggetto. </li><li> Ogni cotiledone si osserva, dalla corticale alla regione mediale, in fluorescenza per ogni distribuzione subcellulare alterata del marcatore organello. </li><li> Impianti positivi vengono trapiantate nel terreno ed i loro semi sono raccolti e selezionati per confermare il fenotipo mutante nella generazione M3. </li><li> Rimuovere i contaminanti mutazioni di fondo attraverso backcrossing almeno tre volte a una wild-type genoma eventualmente containing il marcatore desiderato organello fluorescente. <ol><li> Dalla pianta madre, usando le forbici o pinze sottili rimuovere silique mature e fiori aperti. </li><li> Rimuovere le gemme che sono troppo piccoli da meristema. </li><li> Inserire la punta di una coppia di pinze tra petali e sepali di aprire uno bocciolo di fiore e rimuovere tutte le antere. </li><li> Utilizzando una coppia di pinze prendere un fiore aperto matura dalla pianta padre e strofinare le antere sullo stimma della pianta snervante. </li></ol></li></ol> 3. Mapping Questa sezione descrive essenzialmente come mappare una mutazione recessiva utilizzando un protocollo modificato dal Borevitz 9, che è più veloce rispetto ai metodi tradizionali di mappatura 10,11. Questo approccio utilizza array ad alta densità oligonucleotidici con la capacità di rilevare numerose tecniche singoli polimorfismi (SFP) in un singolo test 12. Utilizzando la Arabidopsis Affymetrix ATH1 serie GeneChip è possibileanalizzare i circa 24.000 geni. Un pool di F 2 individui che mostrano la mutazione viene confrontato con un pool di wild-type F 2 piante raccolte all&#39;interno della stessa popolazione segregante. Poi, la mutazione verrà mappato nella regione in cui si arricchisce il pool mutante per alleli mutanti genotipo e di conseguenza nella stessa regione il wild-type piscina risulterà arricchito per l&#39;alleli wild-type genitori 13. <ol><li> Il DNA genomico (3 mg) viene estratto dal omozigote mutante Columbia (M3) utilizzando un impianto Qiagen DNeasy Mini Kit e viene sottoposto Illumina Genome Analyzer II (GA II) 14 sequenziamento. </li><li> Il omozigote mutante Columbia (M3) è attraversata con Landsberg erecta per generare una popolazione di mappatura. </li><li> La mappatura viene eseguita su 70 fino a 100 individui F2 mostrano il fenotipo aberrante e lo stesso numero di piante F2 con un fenotipo di tipo selvatico. </li><li> Raccogliere da ogni pianta un disco foglia (0,60 mm) utilizzando un punteruolo. Ildisco di foglia devono essere raccolti dalle foglie della stessa età per essere sicuri di avere simili quantità di DNA. I campioni possono essere trattati per l&#39;estrazione genomico separatamente o in gruppi. In questo caso, è possibile raccogliere campioni 5-10 per ogni provetta Eppendorf in modo che alla fine si avrà meno provette Eppendorf da DNA genomico che deve essere estratto. </li><li> Impianto MasterPure Leaf DNA Purification Kit (Epicentre) viene utilizzato per estrarre il DNA genomico. Il DNA genomico ottenuto da ciascun campione viene quantificata con un NanoDrop. </li><li> La stessa quantità di DNA genomico da ciascun campione viene poi messo insieme fino a un totale di 300 ng (~ 30 mL) di DNA vegetale; aggiungere 60 ul 2.5X soluzione primer casuale [125 mM Tris-HCl (pH 6,8), 12,5 mM MgCl2, 25 mM 2-mercaptoetanolo, 750 pg / ml primer oligodeoxyribonucleotide (octamers casuali)] (Bioprime kit) e 42 microlitri di acqua (volume finale 132 pl). </li><li> DNA denaturano a&gt; 95 ° C per 5 a 10 min. </li><li> Raffreddare su ghiaccio. </li><li> Ad ogni denaturosso DNA aggiungere 15 ul miscela dNTP 10 volte con biotina dCTP [1 mM biotina-14-dCTP, dCTP 1 mM, 2 mM dATP, 2 mM dGTP, dTTP 2 mM a 10 mM Tris-HCl (pH 7,5), 1 mM Na2EDTA] (Bioprime kit), e completare con 3 polimerasi Klenow microlitri (kit Bioprime). </li><li> Incubare per una notte a 25 ° C. </li><li> Il giorno seguente, aggiungere 15 microlitri 3 M NaOAc e 400 microlitri freddo EtOH 100%; mix. </li><li> Incubare a -80 ° C per 1 ora, poi girare a 20.500 xg per 15 min, rimuovere supernatante, e lavare con 500 microlitri EtOH freddo 75%. </li><li> Spin a 20.500 xg per 10 min. </li><li> Pellets di DNA a secco a 37 ° C per 10 min e risospendere in 100 microlitri di acqua. </li><li> Utilizzare 5 microlitri per controllare resa e qualità su un gel (Figura 2). </li><li> Invia 95 microlitri di etichettatura di reazione per il wild-type e mutanti per l&#39;ibridazione Arabidopsis Genome Array GeneChip ATH1. </li><li> Gli array vengono scansionati ed i. CEL file ottenuti sono analizzati utilizzando il software R ( <a href="http://www.r-project.org/">http://www.r-project.o</a>rg /). </li><li> Installare il software R, quindi aprire il programma e incollare la seguente stringa: fonte (&quot; <a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a> &quot;) biocLite () Poi premere il tasto-questo ritorno installerà i pacchetti standard (Bioconductor <a href="http://www.bioconductor.org">http://www.bioconductor.org</a> ). </li><li> In una nuova cartella sul desktop, scaricare dal seguente sito Web ( <a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a> ) i file: readcel.R SFP.R Map.R ath1V5.RData ColLerCEL.zip Il ColLerCEL.zip file deve essere decompresso ed i file risultanti incollato nella nuova cartella. </li><li> Rinominare i dati ottenuti dall&#39;esperimento GeneChip in wildtype.CEL e mutant.CEL. </li><li> Copiate ora i tuoi dati GeneChip (wildtype.CEL emutant.CEL) nella cartella. </li><li> Apri R, a) per Mac: fare clic su &quot;Misc&quot; e quindi fare clic su &quot;Cambia directory di lavoro.&quot; b) per PC: fare clic su &quot;File&quot; &quot;Cambia directory&quot; e poi </li><li> a) per Mac: selezionare la cartella contenente i file. Fare clic su &quot;Area di lavoro&quot;, &quot;spazio di lavoro Load File&quot; e selezionare ath1V5.RData, b) per PC: selezionare la cartella contenente i file di cui sopra, cliccare su &quot;File&quot;, poi &quot;Load workspace&quot; e selezionare ath1V5.RData. </li><li> ReadCEL.R Apri utilizzando il Blocco note e copiare l&#39;intero testo di R. </li><li> SFP.R Apri utilizzando il Blocco note e copiare l&#39;intero testo di R. </li><li> Map.R Apri utilizzando il Blocco note e copiare l&#39;intero testo di R. </li><li> Nella finestra della console verrà visualizzato il seguente messaggio: Cromosoma x limita Mb yz I geni della ricerca a TAIR incollare questo link 3 <a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y</a>&amp; End = z </li><li> Copia e incolla il link nel proprio browser internet. Apparirà una finestra e chiedere di aprire o salvare il file, si dovrebbe selezionare Salva. Scegli un nome di file e fissare l&#39;estensione &quot;. Xls&quot;, quindi fare clic su Salva. </li><li> Aprire il file &quot;. Xls&quot; dove si possono trovare le coordinate per il vostro mutante (il risultato è rappresentato graficamente in Figura 3). </li><li> All&#39;interno dell&#39;area mappata nel Illumina assemblato legge del genoma mutante identificare specifiche transizioni EMS quattro tra i polimorfismi a singolo nucleotide. Completano il fenotipo mutante mediante trasformazione con il gene wild type (s) utilizzando le procedure standard 15. </li></ol> 4. Risultati rappresentativi La figura 1 mostra l&#39;approccio utilizzato per l&#39;identificazione di un mutante di percorso secretorio di screening utilizzando la microscopia confocale. Figura 2 mostra una tipica preparazione di DNA genomico marcato per ibridazione matrice. In figura 3, un tipico risultato atteso dopo l&#39;analisi dei dati ottenuti dalla Array GeneChip Arabidopsis Genome ATH1 viene presentato. <img alt="Figura 1" src="/files/ftp_upload/3809/3809fig1.jpg" /> Figura 1. Piante di Arabidopsis transgeniche esprimenti ssGFPHDEL (ER marker) (1) sono stati coltivati ​​per produrre abbastanza semi per EMS trattamento (2). I semi trattati con EMS sono stati poi seminati per generare piante M1 (3). Ogni pianta M1 rappresenta una linea diversa e semi sono stati raccolti separatamente da ciascuno di essi. Seme M2 sono state piastrate su substrato ½ MS (4) e poi sottoposti a screening per difetti di morfologia ER tramite microscopia confocale (5). Durante la proiezione abbiamo trovato piante che conserva la wild-type morfologia ER (6) e le piante che mostrano fenotipi difettosi ER (7). Queste piante sono state coltivate per ottenere la generazione M3 e per confermare il fenotipo (8). Il DNA genomico di pianta M3 è stato utilizzato per Solexa Illumina sequenziamento (a). La stessa piantaè stato utilizzato anche per gli incroci con Ler-peso per ottenere la popolazione F2 mappatura (b). <img alt="Figura 2" src="/files/ftp_upload/3809/3809fig2.jpg" /> Figura 2. Reazioni Bioprime etichettatura random (5 pl di 100 pl) sono stati caricati su gel di agarosio 1%. Lane, uno è da un pool di piante F2 wild-type, e Lane b è da un pool di piante mutanti F2. (Marker è 1 Kb DNA ladder-N3232, da NEB). <img alt="Figura 3" src="/files/ftp_upload/3809/3809fig3.jpg" /> Figura 3. La figura rappresenta un esempio di mappatura delle Col-0 mutazione utilizzando Arabidopsis GeneChip ATH1 Genome Hybridization Array. La mutazione in questo esempio è situato sul cromosoma 1 delimitato, da barre verticali. Le barre orizzontali rappresentano le soglie per il rilevamento.

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Qui descritto un microscopio confocale a base di screening per l&#39;identificazione dei mutanti endomembrane. Questo approccio può essere facilmente estesa ad altri organelli della cella per cui specifici marcatori proteici fluorescenti sono disponibili. Lo schermo si basa sulla individuazione di mutanti che mostrano una distribuzione aberrante del marcatore fluorescente o in organello bersaglio o organelli che non dovrebbero contenere il marcatore. Rispettivamente, questi mutanti rappresentano popolazioni in cui è c...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Ethylmethane solfonato </td><td> Sigma </td><td> M0880 </td></tr><tr><td> NaOH </td><td> JT Baker </td><td> 3722-05 </td></tr><tr><td> Murashige Skoog basali medi w Gamborg vitamine </td><td> Phyto technolog labor </td><td> M404 </td></tr><tr><td> Phytagel </td><td> Sigma </td><td> P8169-1Kg </td></tr><tr><td> RNeasy mini kit </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> Maestro vegetale puro foglia DNA kit di purificazione </td><td> Epicentre </td><td> MPP92100 </td></tr><tr><td> Bioprime DNA sistema di etichettatura </td><td> Invitrogen </td><td> 18094-011 </td></tr><tr><td> Alcohol 200 prove </td><td> Decan laboratori inc. </td><td> 2716 </td></tr><tr><td> NaOAc </td><td> JT Baker </td><td></td></tr><tr><td> Gene Chip ATH1 Arabidopsisarray di genoma </td><td> Affymetrix </td><td> 900385 </td></tr><tr><td> Provette Falcon 50 mL </td><td> corning </td><td> 430290 </td></tr><tr><td> Provette Eppendorf 1,5 mL </td><td></td><td></td></tr><tr><td> Carta da filtro 90 millimetri </td><td> Whatman </td><td> 1001090 </td></tr><tr><td> Bilancia analitica </td><td> Mettler Toledo AB54-S </td><td> na </td></tr><tr><td> Contalitri volumetrico (wave) shaker </td><td> Heidolph Polymax 1040 </td><td> na </td></tr><tr><td> Centrifuga </td><td> Eppendorf 5417-R </td><td> na </td></tr></tbody></table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

Questo protocollo descrive un microscopio a fluorescenza basato su uno screening di piantine di Arabidopsis e descrive come mappare le mutazioni recessive che alterano la distribuzione subcellulare di uno specifico tag marker fluorescente in via secretoria. Arabidopsis è un potente modello biologico per gli studi genetici a causa della sua dimensione del genoma, tempo di generazione, e la conservazione dei meccanismi molecolari tra i regni. La matrice genotipizzazione come un approccio per mappare la mutazione in alternativa al metodo tradizionale basato su marcatori molecolari è vantaggiosa poiché è relativamente veloce e può consentire la mappatura dei diversi mutanti in un lasso di tempo molto breve. Questo metodo consente l&#39;identificazione di proteine ​​che possono influenzare l&#39;integrità di eventuali organello nelle piante. Qui, come esempio, si propone una schermata per mappare geni importanti per l&#39;integrità del reticolo endoplasmatico (ER). Il nostro approccio, tuttavia, può essere facilmente estesa ad altri organelli cellulari vegetali(Si veda per esempio 1,2), e rappresenta quindi un passo importante verso la comprensione delle basi molecolari che regolano le altre strutture subcellulari.

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Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Una ricerca fondamentale in biologia cellulare è quello di definire i meccanismi che sono alla base l&#39;identità degli organelli che rendono le cellule eucariotiche. Qui vi proponiamo un metodo per identificare i geni responsabili per l&#39;integrità morfologica e funzionale degli organelli vegetali usando la microscopia a fluorescenza e di nuova generazione strumenti di sequenziamento.

Microscopia a fluorescenza-Screening e sequenziamento di prossima generazione: Strumenti utili per l&#39;identificazione di geni coinvolti in organelli Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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12.2K Views.  Michigan State University. Una ricerca fondamentale in biologia cellulare è quello di definire i meccanismi che sono alla base l'identità degli organelli che rendono le cellule eucariotiche. Qui vi proponiamo un metodo per identificare i geni responsabili per l'integrità morfologica e funzionale degli organelli vegetali usando la microscopia a fluorescenza e di nuova generazione strumenti di sequenziamento.

Video: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse imaging di mitosi dopo la trasfezione di siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Ricerca

Biologia

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Utilizzo di RNA-mediata strategia di alimentazione di interferenza secondo schermo per geni coinvolti nella regolazione Dimensione del corpo nel nematode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

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 ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

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

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

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

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificazione, sequenziamento di prossima generazione, e DNA genomico mappatura dei siti di integrazione retrovirali

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Tick Microbiome caratterizzazione ordinando Amplicon del rRNA 16S di prossima generazione

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Analisi microbiota utilizzando PCR in due fasi e sequenziamento genico rRNA 16S di nuova generazione

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

Un protocollo di estrazione del DNA delle zanzare a base di perline magnetiche per il sequenziamento di nuova generazione

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.