We thank Dr. Bas van Steensel for providing the DamID mammalian expression vectors. We thank Yale Center for Genome Analysis and the Genomics Core in Yale Stem Cell Center for advice on preparing NGS libraries and implementing high throughput DNA sequencing. This work was supported by the startup funding from Yale School of Medicine, a Scientist Development Grant from American Heart Association (12SDG11630031) and a Seed Grant from Connecticut Innovations, Inc. (13-SCA-YALE-15).

<ol>
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The authors have nothing to disclose.

Whether Dam-tagged proteins retain the functions of endogenous proteins should be examined before a DamID-seq experiment. The subcellular localization of Dam-tagged nuclear envelope proteins should always be determined and compared with that of the endogenous proteins. For studying transcription factors, it is suggested to examine whether the Dam-fusion protein can rescue the functions of the endogenous protein in regulating gene expression. This functional test can be performed in organisms in which knockout mutants of endogenous DNA-binding proteins are available. Because advances in genome engineering have potentially allowed knocking out any endogenous gene of interest, functions of Dam-tagged DNA-binding proteins can be examined in cultured mammalian cells. 
The critical step in this protocol is to successfully fragment the DpnII-digested DamID PCR products to around 200 bp. This step is designed to render the amplified adenine-methylated fragments to a narrow size range for sequencing and to randomize the starting nucleotides of the DNA fragments in a sequencing library. Inefficient fragmentation will leave the majority of the DNA fragments starting with GATC (the 5'-overhang from the second DpnII digestion), and will result in a much lower performance and yield or even a failure in Illumina sequencing. Other DNA fragmentation methods may be used as an alternative approach. 
The resolution of DamID (and DamID-seq described here) is limited by the frequency of GATCs in the genome to be studied. Moreover, even with high throughput sequencing, the genomic localizations of a DNA-binding protein can only be mapped within two consecutive GATCs rather than to the actual DNA-binding sites. 
Despite its limitation, the DamID assay has important advantages. Because DamID does not require highly-specific antibodies, it can be used to detect a subset of nuclear proteins that could be difficult to assay by ChIP (such as the nuclear envelope proteins). To study how these proteins regulate genome functions, it is important to integrate and cross-analyze their genome-wide localization data with the current epigenomic mapping data (such as data from the ENCODE and NIH Roadmap Epigenomics Projects 30,31). The DamID-seq approach provides both higher resolution and higher sensitivity than DamID by microarray and enables detecting differential NL-associations within gene structures 10. A combinatorial analysis of DamID-seq data, ChIP-seq data 32 and gene expression data has identified a class of NL-associated genes with distinct epigenetic and transcriptional features (data not published). 
Another advantage of DamID is that it only requires a small number of cells. In recent years, there has been an explosion in single cell analysis of gene regulation 33,34. Although genome sequence 35, genome-wide gene expression 36 and chromatin conformation 37 can be assayed in a single cell, there has not been an available approach for detecting protein-DNA interactions genome-wide in a single cell. DamID-seq is a highly promising approach for this goal, and may complement the single cell imaging approach in detecting the dynamics of genome-NL interactions 38. One complication is that because the Dam-fusion protein is expressed at a much lower level than the endogenous protein in the DamID assay, it is possible that the Dam-fusion protein may only occupy a subset of genomic binding sites as compared to the endogenous protein. 
DamID assay has mostly been used in cultured animal cells to detect protein-DNA interactions. Notably, developmental biologists have applied this assay in detecting protein-DNA interactions in specific cell types in vivo. For example, Dam-tagged RNA polymerase II was expressed specifically in Drosophila neural stem cells to detect their genome-wide occupancy without cell isolation 39. DamID-seq will be highly useful to study the genome-wide localizations of nuclear envelope proteins, transcription factors and chromatin regulators during development in animal models.

DNA identificazione adenina metiltransferasi (DamID) 1,2 è un metodo per rilevare le interazioni proteina-DNA in vivo ed è un approccio alternativo per immunoprecipitazione della cromatina (ChIP) 3. Esso utilizza una quantità relativamente bassa di cellule e non richiede reticolazione chimica della proteina con DNA o un anticorpo altamente specifico. Quest&#39;ultima è particolarmente utile quando la proteina bersaglio è liberamente o indirettamente associati con il DNA. DamID è stato utilizzato con successo per mappare i siti di legame di una varietà di proteine ​​tra cui proteine ​​dell&#39;involucro nucleare 4-10, cromatina proteine ​​associate 11-13, cromatina modifica enzimi 14, fattori di trascrizione e co-fattori 15-18 e RNAi macchinari 19. Il metodo è applicabile in diversi organismi compreso S. cerevisiae 13, S. pombe 7, C. elegans 9,17, D. melanogaster 5,11,18,20, A. thaliana 21,22 così come topo e cellule umane linee 6,8,10,23,24. Lo sviluppo del test DamID si è basata sulla rilevazione specifica di adenina-metilato frammenti di DNA nelle cellule eucariotiche che la mancanza adenina endogena metilazione 2. Una proteina di fusione espressa, consistente della proteina legante il DNA di interesse e E. DNA coli adenina metiltransferasi (Dam), può metilare la base adenina in sequenze GATC che sono in prossimità spaziale (la maggior parte in modo significativo entro 1 kb e fino a circa 5 kb) per i siti di legame della proteina nel genoma 2. I frammenti di DNA modificati possono essere specificamente amplificati e ibridati microarray per rilevare i siti di legame genomici della proteina di interesse 1,25,26. Questo metodo originale DamID era limitata dalla disponibilità di microarrays e la densità di sonde predeterminati. Abbiamo quindi integrata high throughput sequencing ina DamID 10 e designato il metodo DamID-ss. Il gran numero di breve letture generata da DamID-ss permette la localizzazione precisa delle interazioni proteina-DNA del genoma-larghi. Abbiamo scoperto che DamID-ss fornito una risoluzione più elevata e una gamma dinamica più ampia rispetto DamID mediante microarray per lo studio del genoma nucleare lamina (NL) 10 associazioni. Questo metodo permette una migliore sondaggio associazioni NL all&#39;interno di strutture geniche 10 e facilita il confronto con altri dati di sequenziamento ad alto rendimento, come ChIP-seq e RNA-Seq. Il protocollo DamID-ss qui descritto è stato inizialmente sviluppato per la mappatura del genoma-NL associazioni 10. Abbiamo generato una proteina di fusione per legare il mouse o Lamin B1 umana a E. DNA metiltransferasi coli adenina e testato il protocollo di 3T3 fibroblasti embrionali di topo, topo C2C12 mioblasti 10 e IMR90 fibroblasti polmonari fetali umane (dati non pubblicati). In questo protocollo, iniziamo con constructing vettori ed esprimere proteine ​​di fusione Dam-tethered di infezione lentivirale in cellule di mammifero 24. Successivamente, si descrivono i protocolli dettagliati di amplificare frammenti di DNA adenina-metilato e preparare le librerie di sequenziamento che dovrebbero essere applicabili in altri organismi.

<table>
	<tbody>
		<tr>
			<td>ViraPower Lentiviral Expression Systems</td>
			<td>Life Technologies</td>
			<td>K4950-00, K4960-00, K4970-00, K4975-00, K4980-00, K4985-00, K4990-00, K367-20, K370-20, and K371-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway BP Clonase II Enzyme Mix</td>
			<td>Life Technologies</td>
			<td>11789-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Enzyme Mix</td>
			<td>Life Technologies</td>
			<td>11791-020</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue Kit (250)</td>
			<td>QIAGEN</td>
			<td>69506 or 69504&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway pDONR 201</td>
			<td>Life Technologies</td>
			<td>11798-014</td>
			<td></td>
		</tr>
		<tr>
			<td>293T cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/25300054">Trypsin-EDTA (0.05%), phenol red</a></td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, pyruvate</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>200 Proof EtOH</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs</td>
			<td>R0176</td>
			<td>supplied with buffer</td>
		</tr>
		<tr>
			<td>DamID adaptors &#34;AdRt&#34; and &#34;AdRb&#34;</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>sequences available in ref. 24; no phosphorylation of the 5&#39; or 3&#39; end to prevent self-ligation.</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche Life Science</td>
			<td>10481220001</td>
			<td>supplied with buffer</td>
		</tr>
		<tr>
			<td>DpnII</td>
			<td>New England Biolabs</td>
			<td>R0543</td>
			<td>supplied with buffer</td>
		</tr>
		<tr>
			<td>DamID PCR primer &#34;AdR_PCR&#34;</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>sequences available in ref. 24</td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Set</td>
			<td>New England Biolabs</td>
			<td>N0446</td>
			<td>100 mM each of dATP, dCTP, dGTP and dTTP</td>
		</tr>
		<tr>
			<td>Advantage 2&#160; Polymerase Mix</td>
			<td>Clontech</td>
			<td>639201</td>
			<td>supplied with buffer</td>
		</tr>
		<tr>
			<td>1Kb Plus DNA Ladder</td>
			<td>Life Technologies</td>
			<td>10787018</td>
			<td>1.0 &#181;g/&#181;l</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28104 or 28106</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit</td>
			<td>QIAGEN</td>
			<td>28004 or 28006</td>
			<td>for an elution volume of less than 30 &#181;l</td>
		</tr>
		<tr>
			<td>SPRI beads / Agencourt AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>apply extra mixing and more elution time if less than 40 &#181;l elution buffer is used</td>
		</tr>
		<tr>
			<td>Buffer EB</td>
			<td>QIAGEN</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext dsDNA Fragmentase</td>
			<td>New England Biolabs</td>
			<td>M0348</td>
			<td>supplied with buffer</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Reaction Buffer</td>
			<td>New England Biolabs</td>
			<td>B0202</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Polymerase I, Large (Klenow) Fragment</td>
			<td>New England Biolabs</td>
			<td>M0210</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynuleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow Fragment (3&#8217; -&#62; 5&#8217; exo-)</td>
			<td>New England Biolabs</td>
			<td>M0212</td>
			<td>supplied with buffer</td>
		</tr>
		<tr>
			<td>sequencing adaptors</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>sequences available in ref. 28</td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>New England Biolabs</td>
			<td>M2200</td>
			<td>used in 11.2; supplied with Quick Ligation Reaction Buffer and Quick T4 DNA Ligase</td>
		</tr>
		<tr>
			<td>sequencing primer 1 and 2</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>sequences available in ref. 28</td>
		</tr>
		<tr>
			<td>KAPA HiFi PCR Kit</td>
			<td>Kapa Biosystems</td>
			<td>KK2101 or KK2102</td>
			<td>supplied with KAPA HiFi DNA Polymerase, 5X KAPA HiFi Fidelity Buffer and 10mM dNTP mix</td>
		</tr>
		<tr>
			<td>agarose</td>
			<td>Sigma Aldrich</td>
			<td>A4679</td>
			<td></td>
		</tr>
		<tr>
			<td>ethidium bromide</td>
			<td>Sigma Aldrich</td>
			<td>E1510-10ML</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>QIAGEN</td>
			<td>28704 or 28706</td>
			<td></td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Bio-Rad Laboratories</td>
			<td>1725121 or 1725122</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>0.45 um PVDF Filter</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>25 ml Seringe</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>10 cm Tissue Culture Plates</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>6-well Tissue Culture Plates</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>S1000 Thermal Cycler</td>
			<td>Bio-Rad Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories</td>
			<td></td>
			<td>for qPCR</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Dry Block Heater or Thermomixer</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Gel electrophoresis system with power supply</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>Magnet stand</td>
			<td></td>
			<td></td>
			<td>for purification of DNA with SPRI beads; should hold 1.5-2 ml tubes; brand not critical</td>
		</tr>
		<tr>
			<td>UV transilluminator</td>
			<td></td>
			<td></td>
			<td>brand not critical</td>
		</tr>
		<tr>
			<td>E-gel electrophoresis system</td>
			<td>Life Technologies</td>
			<td>G6400, G6500, G6512ST</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La proteina di fusione Dam-V5-LmnB1 è stato verificato per essere co-localizzato con il endogena Lamin B proteine ​​mediante immunofluorescenza (Figura 1). Il successo di amplificazione PCR di frammenti di DNA adenina-metilato è un passo fondamentale per DamID-ss. I campioni sperimentali dovrebbero amplificare una macchia di 0,2 - 2 kb, mentre i controlli negativi (senza DpnI, senza ligasi o senza mascherina PCR) dovrebbe portare a non-o chiaramente meno-amplificazione (Figura 2). I frammenti di DNA metilati sono nell&#39;intervallo da 0,2 a 2 kb, mentre la dimensione dell&#39;inserto desiderata per una libreria NGS è da 200 a 300 bp. Pertanto, è essenziale per frammentare i prodotti di PCR di metile nella gamma formato adatto. Tuttavia, è stato trovato per essere poco pratico per rompere contemporaneamente frammenti di DNA più grandi a dimensioni adattes e mantenere la maggior parte dei piccoli frammenti di DNA intatto in una singola durata frammentazione. Pertanto, gli esperimenti sono stati eseguiti corso di tempo per determinare il tempo minimo (T 0.2kb) necessario per frammento di 1 ug di DNA ad uno striscio centrato a 200 bp (Figura 3). Poi sono stati selezionati 6 durate di tempo in incrementi uguali tra 5 minuti e T 0.2kb per la frammentazione attuale. L&#39;attività enzimatica della doppia elica di DNA Fragmentase può variare da lotto a lotto e può diminuire nel tempo, quindi si consiglia di ripetere questa operazione per un nuovo lotto di Fragmentase o dopo conservazione per un periodo di tempo. La dimensione inserto desiderato è compreso tra 200 e 300 bp corrispondente a frammenti di DNA tra 300 e 400 bp (compresi 121 bp adattatori sequenziamento) sul gel di agarosio. Tre fette sottili all&#39;interno di questa gamma sono stati asportati da ciascun campione sperimentale per restringere la gamma di dimensioni di una biblioteca e di aumentare la possibilitàdi ottenere almeno una libreria sequenziamento qualificato (Figura 4). Un&#39;aliquota di 5 ml di ogni biblioteca DNA amplificato è stato analizzato il gel per determinare quale biblioteca possono qualificarsi per il sequenziamento. Come mostrato in Figura 5A, una chiara singola banda della stessa dimensione della fetta gel escisse dovrebbe essere visibile sul gel di agarosio (fase 3.7.4). Avanti, librerie selezionate sono stati esaminati da un Bioanalyzer (Figura 5B) per determinare la gamma esatta dimensione e concentrazione prima di sequenziamento. Se lo si desidera, le biblioteche di DNA amplificati possono essere esaminati direttamente da un Bioanalyzer senza l&#39;analisi di gel. Quando più librerie sono di buona qualità, si consiglia di sequenziare librerie di dimensioni simili gamme per un paio di sperimentali (cellule che esprimono Dam-V5-POI) e di controllo (cellule che esprimono V5-Dam) campioni. Il corto si legge generato dasistemi di sequenziamento sono stati mappati torna alla corrispondente genoma. Unicamente allineato letture venivano poi passati alle analisi successive. Una conduttura di elaborare a breve legge, costruire un&#39;interazione mappa del genoma-NL e analizzare le associazioni gene-NL sono stati descritti in dettaglio nel nostro precedente lavoro 10. Risultati rappresentativi sono mostrati in Figura 6. <img alt="Figura 1" src="/files/ftp_upload/53620/53620fig1.jpg" /> Figura 1. Convalida localizzazioni subnucleari di proteine ​​di fusione da immunofluorescenza. IMR90 cellule di fibroblasti polmonari umane sono state transitoriamente trasfettate con un plasmide che esprime Dam-V5-LmnB1 e sono state colorate da anti-Lamin B (A, rossa) e anti-V5 (B, verde). (C) Unire di immagini in A e B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig1large.jpg" target="_blank">Si prega di cliccare qui per vedere una versione più grande di t</a>la sua figura. <img alt="Figura 2" src="/files/ftp_upload/53620/53620fig2.jpg" /> Figura 2. PCR amplificare frammenti di DNA adenina-metilato. Un&#39;aliquota di 5 microlitri da ogni reazione PCR è stata analizzata su gel di agarosio all&#39;1%. Uno striscio che vanno 0,2-2 kb è stato amplificato da ogni campione sperimentale, ma nessuna amplificazione è stata osservata nei controlli negativi (nessun DpnI al punto 2.1, non Ligase al punto 2.2, o nessun modello PCR). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53620/53620fig3.jpg" /> Figura 3. Determinazione durate ottimali di frammentazione. I prodotti di PCR purificati metile sono stati sottoposti a frammentazione per durate di tempo 5-55 min a un incremento di 10 min (DNA non digerito<html> , Etichettato come &quot;0 min&quot;). 1 pg di DNA ladder e 0,5 mg di ciascun campione di DNA frammentato sono stati analizzati su un gel di agarosio. Il tempo minimo per digerire la maggioranza della striscio DNA di circa 200 bp è stato determinato in circa 35 minuti, quindi sei durate di tempo equidistanti tra 5 e 35 min (5 e 35 min incluse) sono stati selezionati per eseguire la frammentazione attuale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig3large.jpg" target="_blank">Favore clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53620/53620fig4.jpg" /> Figura 4. Formato selezionando le librerie di DNA. Campioni di DNA di Dam-V5-LmnB1 e V5-Dam dal punto 3.6 sono stati eseguiti su un gel di agarosio al 2%, e tre fette di gel uguali dimensioni (L, M e H corrispondente a bassa, media ed alta dimensioni) venivano asportate tra 300 e 400 bp come mostrato dalle linee gialle.<html> om / files / ftp_upload / 53620 / 53620fig4large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="Figura 5" src="/files/ftp_upload/53620/53620fig5.jpg" /> Figura 5. librerie di DNA amplificato per alta sequenziamento rendimento. (A) amplificata librerie di DNA analizzati su un gel di agarosio. Modelli di PCR sono stati purificati da fette di gel mostrato in figura 4. (B) Bioanalyzer risultati del campione V5-Dam (L) e il campione Dam-V5-LmnB1 (L) che sono stati sottolineato in (A). Questi due biblioteche avevano gli intervalli di dimensioni simili stretto e quindi qualificato per alta sequenziamento rendimento. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig5large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> jpg &quot;/&gt; Figura 6. dati NGS visualizzati nel UCSC browser genomico. Cromosoma mouse 1 Di seguito riportiamo come esempio. Dati della sequenza sono state prodotte da mioblasti topo C2C12 10. Tracce &quot;MB.LmnB1.w2k&quot; e &quot;MB.Dam.w2k&quot;, corrispondenti ai dati provenienti da cellule che esprimono rispettivamente Dam-V5-LmnB1 e V5-Dam, trama normalizzato densità di lettura (legge per kilobase per milione mappata univocamente legge, o RPKM ) a non sovrapposte consecutivi finestre 2 kb lungo il cromosoma. Tieni traccia trame &quot;MB.log2FC.w2k&quot; associazioni genoma-NL, cioè log2 RPKM rapporti di LmnB1 oltre Dam, a 2 risoluzione kb. Traccia &quot;MB.sLADs&quot; dipinge Lamina Associated Domini basato sequenziamento-(slads, ossia regioni genomiche che hanno densità di lettura più elevate di LmnB1 su Dam con significatività statistica) in nero, non slads nelle regioni grigie e indeterminati in bianco. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig6large.jpg" target="_blank">Cliccate qui per downloa</a>d questo file.

Watch this Scientific Journal Video about DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments at JoVE.com

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

Descriviamo qui un test con l&#39;accoppiamento di identificazione del DNA adenina metiltransferasi (DamID) ad alto rendimento sequenziamento (DamID-ss). Questo metodo fornisce una migliore risoluzione più elevata e una gamma dinamica più ampia, e permette di analizzare i dati DamID-Seq in combinazione con altri dati di sequenziamento ad alto rendimento, come 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 ...

damid-seq-genome-wide-mapping-protein-dna-interactions-high

Nanyang Technological University

Research

JoVE Journal

Biology

19.3K Views.  Yale University School of Medicine.  Descriviamo qui un test con l'accoppiamento di identificazione del DNA adenina metiltransferasi (DamID) ad alto rendimento sequenziamento (DamID-ss). Questo metodo fornisce una migliore risoluzione più elevata e una gamma dinamica più ampia, e permette di analizzare i dati DamID-Seq in combinazione con altri dati di sequenziamento ad alto rendimento, come ChIP-seq, RNA-Seq, etc. 

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

Electroeluting DNA Fragments

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.

This procedure allows the purification of DNA fragments with high yield.

Electroeluting frammenti di DNA

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a ...

Ricerca

Biologia

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitative e automatica High-throughput Genome-wide Schermi RNAi in C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

Elettroforesi su gel agarosio per la separazione di frammenti di DNA

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genome-wide gene delezioni in<em&gt; Streptococcus sanguinis</em&gt; Da Throughput PCR ad alta

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

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

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

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 ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

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 ...

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 ...