Il lavoro è supportato da un Premio Istituto per lo sviluppo da Ospedale Pediatrico di Filadelfia.

1. Cross-Link cromatina <ol><li> Crescere le cellule in piatti di coltura di cellule 100x20mm. La quantità di celle può variare da 1 a 10 milioni di cellule per piastra seconda del tipo cellulare. Circa 2 milioni di cellule è sufficiente per una immunoprecipitazione. </li><li> Cellule del legame incrociato a 1% di formaldeide per 10 minuti a temperatura ambiente con dondolo occasionale. </li><li> Quench reticolazione aggiungendo una concentrazione finale di 125 mM glicina e incubare per 5 min a temperatura ambiente. </li><li> Lavare le cellule con fosfato 1X Soluzione salina tamponata (PBS) due volte, decantare PBS e aggiungere 0,2 ml di PBS. </li><li> Celle di raccolta con un raschietto cellula di plastica in una provetta. </li><li> Centrifugare le cellule a 2000 rpm per 5 minuti a 4 ° C. </li><li> Aspirare il surnatante. Risospendere le cellule in SDS tampone di lisi (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1) per l&#39;intero lisato o tenerli come pellet per l&#39;estrazione nucleare. </li><li> Le cellule possono essere salvati a -80 ° C oppure si può procedere immediately con sonicazione. </li></ol> 2. Preparare Nuclei (Andare al punto 3.5 per intero lisato) <ol><li> Supplemento Cell Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) con 1X proteinasi ogni esperimento. </li><li> Risospendere scongelati pellet cellulare in circa 10 volte il volume di pellet con Cell Lysis Buffer. </li><li> Dounce-omogeneizzare 10 volte con pestello poi incubare in ghiaccio per 10 min. </li><li> Centrifugare campione a 4000 rpm per 5 minuti a 4 ° C, scartare il surnatante, e salvare pellet nucleare. </li></ol> 3. Sonicazione * <ol><li> Warm up SDS tampone di lisi e la quantità supplemento di tampone per essere utilizzato con proteinasi. </li><li> Risospendere il pellet nucleare in SDS Lysis Buffer (circa 0,5 ml di tampone per 1-10.000.000 cellule) </li><li> Incubare in ghiaccio per 10 min. </li><li> Aggiungere aliquote da 0,5 ml di campioni per provette da microcentrifuga. </li><li> Sonicare su ghiaccio bagnato utilizzando Misonix sonicator con 30 sec e 45 sec suoff in un ambiente ampiezza 2. Numero di cicli per dimensione ideale frammento può essere determinata prima provando varie numeri del ciclo (es. 2, 4, 8, 12, 16, e 20 o più cicli). Una diversa marca di sonicazione può essere utilizzato, tuttavia, le condizioni varieranno. La sperimentazione con il numero di cicli e la quantità di tempo e spegnimento devono essere eseguite per determinare le condizioni ideali. </li><li> Raccogliere 20 ml di ogni campione per verificare i risultati sonicazione e di fare la quantificazione,. Il resto del campione può essere conservato a -80 ° C. </li><li> Diluire il 20 l di campione con l&#39;aggiunta di 30 ml di 0,1 X. tampone TE. </li><li> Trattare campione con 1 ml di RNasi A a 37 ° C per 1 ora si aggiunge 1 ml di proteinasi K e incubare a 62 ° C per 2 h. </li><li> Eseguire 20 microlitri del campione su un gel di agarosio al 2%. </li><li> Purificare l&#39;importo residuo del campione con il kit di purificazione PCR QIAquick poi quantificare con spettrofotometro NanoDrop. </li></ol> * Per i nativi CHIP, microccocal nucleasi digestione può essere usato in alternativa al taglio del DNA. 4. Block Beads agarosio * <ol><li> Se perle sono già bloccati, procedere al punto 5.1. </li><li> Utilizzare proteina A o proteina G agarosio. Per 5 immunoprecipitazione (IP), utilizzare 600 ml di 50% tallone liquami (300 ml tallone pellet) </li><li> Per lavare le perline, centrifugare giù a 800 rpm per 1 min a 4 ° C e scartare il surnatante. Aggiungere un po &#39;più di 2 ml di ChIP diluizione (0,01% SDS, 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 16,7 mM Tris-HCl pH 8.1) e mescolare lentamente capovolgendo la provetta 10 volte. Spin giù di nuovo a 800 rpm per 1 min a 4 ° C e scartare il surnatante. Ripetere il lavaggio altre due volte. </li><li> Bloccare perline ruotando a 4 ° C per una notte in una soluzione bloccante. Per la ricetta della soluzione di blocco, fare riferimento alla Tabella 1. </li></ol> 5. Pre-chiaro della cromatina <ol><li> Thaw sonicata cromatina sul ghiaccio. </li><li> Centrifugare a 12.000 rpm fo 10 minuti a 4 ° C poi messo in ghiaccio immediatamente per rimuovere SDS (bianco pellet). </li><li> Raccogliere il surnatante, scartare pellet, e combinare i campioni, se necessario. </li><li> Estrarre gli importi necessari per l&#39;esperimento sulla base di calcoli (1-10 ug della cromatina per IP). </li><li> Diluire cromatina 10X in ChIP tampone di diluizione integrato con inibitore della proteasi. </li><li> Aggiungere 100 ml di perline bloccate per IP. </li><li> Ruota a 4 ° C per 1 ora. </li></ol> 6. Immunoprecipitation <ol><li> Centrifugare i campioni a 800 rpm per 1 min e trasferire il surnatante in una nuova provetta. </li><li> Spin down surnatante a 800 rpm per 1 min e trasferimento in un&#39;altra provetta pulita. </li><li> Risparmiare 20 pl del supernatante, per servire come controllo di input, a -20 ° C. </li><li> Aliquota della cromatina per il numero di indirizzi IP da fare nell&#39;esperimento. </li><li> Aggiungere 2 ug di anticorpo per 1-10 ug di cromatina per ogni campione. </li><li> Incubare per una notte a 4 ° C con rotazione. </li><li> Aggiungere 100 ml di perline bloccate per ogni campione IP. </li><li> Incubare per 1 ora a 4 ° C con rotazione. </li><li> Pellet le perline riducendo la velocità a 800 rpm per 1 min e scartare la maggior quantità di surnatante possibile. </li><li> Lavare perle di una volta con sale tampone di lavaggio Complesso immunitario basso. Aggiungere 1 ml di tampone a ciascuna provetta; ruotare a temperatura ambiente per 5-8 min; centrifugare a 800 rpm per 1 minuto, quindi eliminare il surnatante. Ripetere il lavaggio una volta con Immune tampone di lavaggio ad alta Salt Complex e tampone di lavaggio complesso immune LiCl e due volte con tampone TE per un totale di 5 lavaggi (Tabella 2). </li></ol> 7. Eluizione <ol><li> Campioni di ingresso disgelo da giorno precedente per essere elaborati con i eluenti. </li><li> Fai tampone di eluizione fresco (Tabella 3). </li><li> Fare un master mix di abbastanza tampone di eluizione necessaria per IP e campioni di controllo di ingresso più 1-2 campioni extra. </li><li> Aggiungere 100 microlitri di buffer di eluizione per ogni campione IP e incubare a temperatura ambiente per 15 mcon rotazione. </li><li> Centrifugare a 800 rpm per 1 min e aggiungere surnatante in una nuova provetta. </li><li> Aggiungere altri 100 ml di buffer di eluizione a ciascuna provetta di perline e incubare a temperatura della camera per 15 minuti con rotazione. </li><li> Vortex per 15 secondi dopo l&#39;incubazione, centrifugare a 5000 rpm per 1 minuto, quindi unire il surnatante con il surnatante dalla prima eluizione. (Assicurarsi che non vi siano rimasti più di perline nel surnatante. Se non sei sicuro, spin down surnatante di nuovo a 5.000 rpm per 1 minuto e raccogliere il surnatante in una nuova provetta. </li><li> Aggiungere 180 ml di tampone di eluizione al 20 l di campioni di controllo di input. </li></ol> 8. Reverse Cross-Link <ol><li> Al 200 pl di eluenti e controlli di input, aggiungere 8 ml di NaCl 5 M. </li><li> Tubi di sigillare con parafilm e incubare in bagnomaria a 65 ° C durante la notte. </li></ol> 9. Purificazione del DNA <ol><li> Trattare ogni campione con 1 ml di RNasi A per 1 ora a 37 ° C. </li><li> Aggiungere 4 μl di 0,5 M EDTA, 8 microlitri 1M Tris-HCl, miscela, si aggiunge 1 ml di proteinasi K per ogni campione e incubare a 45 ° C per 2 h. </li><li> Purificare i campioni utilizzando kit di purificazione PCR QIAquick. I campioni possono essere conservati a -20 ° C e controllo PCR può essere fatto in una data successiva. </li></ol> * In alternativa, perle magnetiche ChIP-grado può essere usato al posto di agarosio per la porzione immunoprecipitazione. 10. Controllo PCR <ol><li> Per il controllo di PCR, utilizzare primer per regioni note per essere vincolati dalla proteina di interesse. Inoltre, utilizzare primer per le regioni non vincolanti come controlli negativi. </li><li> Miscelare i reagenti per la reazione. Diluire il campione di ingresso a 1:100 (Tabella 3). </li><li> Esegui reazione. Programma di PCR: </li></ol> Passo 1: 94 ° C 3 min Passo 2: 94 ° C 20 sec 59 ° C 30 sec 72 ° C 30 sec (Ripetere il passaggio 2 per almeno 30 cycles) Fase 3: 72 ° C 2 min <ol start="4"><li> Analizzare i campioni su 1% gel. </li><li> L&#39;arricchimento può anche essere determinato quantitativamente con real-time PCR. </li></ol>

<ol>
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Gli autori dichiarano di non avere interessi finanziari in competizione.

E &#39;ora possibile effettuare un genoma profilo di proteina-DNA associazione interazioni utilizzando ChIP-Seq, come è stato di recente dimostrato con altri fattori di trascrizione 2,3. La chiave per un esito sequenziamento di successo è la generazione di un modello di alta qualità cromatina immunoprecipitazione DNA. Una volta che il DNA templato è stato generato e accertato essere opportunamente arricchito, si può poi prendere in preparazione per la successiva raccolta sequ...

Per molti anni c&#39;è stato un bisogno insoddisfatto per identificare l&#39;insieme di geni legati e regolata da una proteina determinato genoma, in particolare quelli della classe fattore di trascrizione. Odom et al. 1 usato immunoprecipitazione della cromatina (ChIP) in combinazione con microarrays promotore per identificare sistematicamente i geni occupati dai regolatori trascrizionali pre-specificati, in fegato umano e di isole pancreatiche. Successivamente, Johnson et al. 2 sviluppato un saggio di immunoprecipitazione della cromatina larga scala basata sul sequenziamento del DNA ultra high throughput diretto (ChIP-seq) per mappare completo interazioni proteina-DNA in interi genomi dei mammiferi. Come banco di prova, hanno mappato in vivo il legame del fattore silenziatore neurone-restrittivo (NRSF) a 1946 posizioni nel genoma umano. I dati visualizzati forte risoluzione di posizione di rilegatura (+ 50 paia di basi), che ha facilitato sia l&#39;isolation di motivi e l&#39;identificazione di motivi NRSF vincolanti. Questi dati di ChIP-Seq avevano anche elevata sensibilità e specificità e confidenza statistica (P &lt;10 -4), proprietà che sono importanti per inferire nuove interazioni candidati. Robertson et al. 3 utilizzato anche ChIP-seq, al fine di mappare STAT1 obiettivi in interferone-γ (IFN-γ)-stimolato e non stimolate le cellule HeLa S3 umane in vivo. Da Chip-Seq, con 15,1 e 12,9 milioni di sequenza mappata univocamente legge, e un tasso stimato falsa scoperta di meno di 0.001, hanno identificato 41.582 e 11.004 putative regioni STAT1 vincolanti in cellule stimolate e non stimolate, rispettivamente. Dei 34 loci noti per contenere STAT1 interferone-reattiva siti 4-8 vincolante, ChIP-seq trovato 24 (71%). Obiettivi di ChIP-Seq sono stati arricchiti in sequenze simili a note STAT1 motivi di legame. Confronti con due dati di ChIP-PCR esistenti set suggeritiche la sensibilità ChIP-seq era tra il 70% e il 92% e la specificità era almeno il 95%. Inoltre, era chiaro che ChIP-seq offre sia a bassa complessità analitica e la sensibilità che aumenta con la profondità di sequenziamento. Come tecnologie come, del genoma &quot;nuova generazione&quot; di sequenziamento forniscono 1-2 ordini di grandezza della quantità di sequenza che può essere costo-efficace generato più vecchie tecnologie 9. Metodi di ChIP-Seq quindi forniscono direttamente la copertura dell&#39;intero genoma per un efficace profiling delle interazioni proteina-DNA di mammifero 3. Nel 2006, una forte associazione di varianti del fattore di trascrizione 7-like 2 (TCF7L2) gene con diabete di tipo 2 è stato scoperto 10. Altri ricercatori hanno già replicato indipendentemente questo risultato in diverse etnie e, curiosamente, dai primi studi di associazione genome-wide di diabete di tipo 2 pubblicati su Nature 11,12 </sup&gt;, Scienza 13-15 e altrove 16,17, l&#39;associazione più forte era effettivamente con TCF7L2, questo è ormai considerata la più significativa scoperta genetica nel diabete di tipo 2 a data 18-20. Inoltre, TCF7L2 è stato collegato al rischio di cancro 21,22, anzi, questa connessione è diventato più evidente quando il locus 8q24 rivelato da genoma ampi studi di associazione di un certo numero di tumori, tra carcinomi colorettali, ha dimostrato di essere dovuto ad un estremo a monte elemento TCF7L2 vincolante guida la trascrizione di MYC 23,24. Come tale, non vi è un grande interesse per la determinazione dei geni a valle regolati da questo fattore di trascrizione chiave. Sulla base dell&#39;esperienza con TCF7L2 come un esempio della metodologia, questo documento descrive come generare modello di DNA chip ad alta qualità. Chip è stato effettuato nella linea cellulare di carcinoma colorettale, HCT116, per la successiva sequenza per costruire un alto risolution mappa dei geni legati da TCF7L2 25 nel tentativo di dare una visione più completa per il suo ruolo chiave nella patogenesi di tratti complessi.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28104 </td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-ChIP Kit </td>
 <td>Millipore </td>
 <td>17-371 </td>
 <td></td>
 </tr>
 <tr>
 <td>GoTaq Hot Start Polymerase </td>
 <td>Promega</td>
 <td>M5001</td>
 <td></td>
 </tr>
 <tr>
 <td>Misonix Sonicator</td>
 <td>Qsonica </td>
 <td>XL-2000 </td>
 <td></td>
 </tr>
 <tr>
 <td>
 NanoDrop 1000 Spectrophotometer </td>
 <td>
 Thermo-Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Positive control primer sequences (TCF7L2-1) 
 Forward- 5'-TCGCCCTGTCAATAATCTCC-3' 
 Reverse- 5'-GCTCACCTCCTGTATCTTCG-3' 
 Negative control primer sequences (CTRL-1) 
 Forward-5'-ATGTGGTGTGGCTGTGATGGGAAC-3'
 
 Reverse- 5'-CGAGCAATCGGTAAATAGGTCTGG-3'</td>
 </tr>
 </tbody>
</table>

generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)

(ChIP-seq) metodi di ChIP-sequencing offrono direttamente la copertura dell&#39;intero genoma, in cui combina immunoprecipitazione della cromatina (ChIP) e il sequenziamento massivamente parallelo può essere utilizzato per identificare il repertorio di sequenze di DNA di mammifero vincolati da fattori di trascrizione in vivo. &quot;Next-generation&quot; tecnologie di sequenziamento del genoma forniscono 1-2 ordini di grandezza della quantità di sequenza che può essere costo-efficace generato più vecchie tecnologie consentendo metodi di ChIP-Seq per fornire direttamente la copertura dell&#39;intero genoma per un efficace profiling dei mammiferi interazioni proteina-DNA. Per successo approcci ChIP-Seq, si deve generare DNA stampo circuito integrato di alta qualità per ottenere i migliori risultati di sequenziamento. La descrizione è basata attorno esperienza con il prodotto proteico del gene più fortemente implicato nella patogenesi del diabete di tipo 2, cioè il fattore di trascrizione fattore di trascrizione 7-like 2 (TCF7L2). Questo fattore è stato anche implicato in vari tumori. Delineato è come generare modello di DNA circuito integrato di alta qualità derivato dalla linea cellulare di carcinoma del colon-retto, HCT116, al fine di costruire una mappa ad alta risoluzione attraverso il sequenziamento di determinare i geni legati da TCF7L2, dando una visione più completa per il suo ruolo chiave nella patogenesi di tratti complessi.

Una volta che la cromatina è stato sonicato e sono stati trattati con RNasi e proteinasi, i campioni eseguiti sul gel di agarosio 2% dovrebbero presentare uno striscio con il grosso del DNA alla dimensione voluta. Se più cicli differenti vengono testati, una graduale diminuzione della dimensione dovrebbe essere visto come il numero di cicli di aumento (Figura 2). Dopo aver completato la porzione immunoprecipitazione del protocollo di arricchimento può sia essere controlla...

Watch this Scientific Journal Video about Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq at JoVE.com

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

La combinazione di immunoprecipitazione della cromatina e sequenziamento ultra-high-throughput (ChIP-seq) in grado di identificare e mappare le interazioni proteina-DNA in una determinata linea di tessuti o cellule. Delineato è come generare un modello di chip per la successiva sequenza di alta qualità, utilizzando l&#39;esperienza con il fattore di trascrizione TCF7L2 come esempio.

Generazione di immunoprecipitazione della cromatina Template DNA di alta qualità per high-throughput sequencing (ChIP-Seq)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

generation-high-quality-chromatin-immunoprecipitation-dna-template

Research

JoVE Journal

Biology

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

24.3K Views.  Children's Hospital of Philadelphia Research Institute. La combinazione di immunoprecipitazione della cromatina e sequenziamento ultra-high-throughput (ChIP-seq) in grado di identificare e mappare le interazioni proteina-DNA in una determinata linea di tessuti o cellule. Delineato è come generare un modello di chip per la successiva sequenza di alta qualità, utilizzando l'esperienza con il fattore di trascrizione TCF7L2 come esempio.

Video: Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Legatura altamente efficiente delle piccole molecole di RNA per microRNA Quantificazione mediante sequenziamento High-Throughput

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

Ricerca

Biologia

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 ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-seq: Genome-wide Mappatura delle interazioni proteina-DNA da High Throughput Sequencing di Adenina-metilato frammenti di DNA

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

High-throughput CRISPR vettore Costruzione e caratterizzazione di modifiche del DNA da Generazione di Roots pomodoro Peloso

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

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

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

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

Un protocollo per la valutazione funzionale del Whole-Protein Saturazione mutagenesi biblioteche Utilizzando High-Throughput Sequencing

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

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Analisi cinetica ad alta velocità ad alto rendimento parallela per cleavage del DNA specifico del sito

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

Metodi modificati per il caricamento di piastre di estrazione del DNA ad alta velocità effettiva riducono il potenziale di contaminazione

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...