Name: damid-seq: genome-wide mapping of protein-dna interactions by high throughput sequencing of adenine-methylated dna fragments
Uploaded: 2016-01-27T18:00:00
Description: 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

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).

1. Nesil ve Fusion Proteinler ve Serbest Barajı Proteinlerin İfadesi <ol><li> DamID vektörüne ilgi klon proteini. <ol><li> Üreticinin protokolüne göre (POI) protein istenen yüksek doğrulukta bir DNA polimerazı kullanılarak, uygun primerler ait cDNA yı çoğaltmak. Deneysel insertlerin düzgün amplifikasyonu sağlamak için uygun amplifikasyon koşulları belirlemek. </li><li> Bir Agaroz ve üreticinin protokolüne uygun olarak jel ekstraksiyon kiti ile özel hedefler amplifıye cDNA arın...

<ol>
	<li>van Steensel, B., Delrow, J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+profiling+using+targeted+DNA+adenine+methyltransferase.">Chromatin profiling using targeted DNA adenine methyltransferase.</a> Nat Genet. 27, 304-308 (2001).</li><li>van Steensel, B., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+in+vivo+DNA+targets+of+chromatin+proteins+using+tethered+dam+methyltransferase.">Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase.</a> Nat Biotechnol. 18, 424-428 (2000).</li><li>Fu, A. Q., Adryan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scoring+overlapping+and+adjacent+signals+from+genome-wide+ChIP+and+DamID+assays.">Scoring overlapping and adjacent signals from genome-wide ChIP and DamID assays.</a> Mol Biosyst. 5, 1429-1438 (2009).</li><li>Guelen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain+organization+of+human+chromosomes+revealed+by+mapping+of+nuclear+lamina+interactions.">Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions.</a> Nature. 453, 948-951 (2008).</li><li>Kalverda, B., Pickersgill, H., Shloma, V. V., Fornerod, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoporins+directly+stimulate+expression+of+developmental+and+cell-cycle+genes+inside+the+nucleoplasm.">Nucleoporins directly stimulate expression of developmental and cell-cycle genes inside the nucleoplasm.</a> Cell. 140, 360-371 (2010).</li><li>Kubben, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+lamin+A-+and+progerin-interacting+genome+regions.">Mapping of lamin A- and progerin-interacting genome regions.</a> Chromosoma. 121, 447-464 (2012).</li><li>Steglich, B., Filion, G. J., van Steensel, B., Ekwall, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inner+nuclear+membrane+proteins+Man1+and+Ima1+link+to+two+different+types+of+chromatin+at+the+nuclear+periphery+in+S.+pombe.">The inner nuclear membrane proteins Man1 and Ima1 link to two different types of chromatin at the nuclear periphery in S. pombe.</a> Nucleus. 3, 77-87 (2012).</li><li>Harr, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+targeting+of+chromatin+to+the+nuclear+lamina+is+mediated+by+chromatin+state+and+A-type+lamins.">Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins.</a> J Cell Biol. 208, 33-52 (2015).</li><li>Gonzalez-Aguilera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+links+emerin+to+neuromuscular+junction+activity+in+Caenorhabditis+elegans.">Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans.</a> Genome Biol. 15, R21 (2014).</li><li>Wu, F., Yao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+compartmentalization+at+the+nuclear+periphery+characterized+by+genome-wide+mapping.">Spatial compartmentalization at the nuclear periphery characterized by genome-wide mapping.</a> BMC Genomics. 14, 591 (2013).</li><li>Filion, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+protein+location+mapping+reveals+five+principal+chromatin+types+in+Drosophila+cells.">Systematic protein location mapping reveals five principal chromatin types in Drosophila cells.</a> Cell. 143, 212-224 (2010).</li><li>Vogel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+heterochromatin+proteins+form+large+domains+containing+KRAB-ZNF+genes.">Human heterochromatin proteins form large domains containing KRAB-ZNF genes.</a> Genome Res. 16, 1493-1504 (2006).</li><li>Venkatasubrahmanyam, S., Hwang, W. W., Meneghini, M. D., Tong, A. H., Madhani, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide,+as+opposed+to+local,+antisilencing+is+mediated+redundantly+by+the+euchromatic+factors+Set1+and+H2A.Z.">Genome-wide, as opposed to local, antisilencing is mediated redundantly by the euchromatic factors Set1 and H2A.Z.</a> Proc Natl Acad Sci U S A. 104, 16609-16614 (2007).</li><li>Shimbo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MBD3+localizes+at+promoters,+gene+bodies+and+enhancers+of+active+genes.">MBD3 localizes at promoters, gene bodies and enhancers of active genes.</a> PLoS Genet. 9, e1004028 (2013).</li><li>Orian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+binding+by+the+Drosophila+Myc,+Max,+Mad/Mnt+transcription+factor+network.">Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network.</a> Genes Dev. 17, 1101-1114 (2003).</li><li>Artegiani, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tox:+a+multifunctional+transcription+factor+and+novel+regulator+of+mammalian+corticogenesis.">Tox: a multifunctional transcription factor and novel regulator of mammalian corticogenesis.</a> EMBO J. , (2014).</li><li>Schuster, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DamID+in+C.+elegans+reveals+longevity-associated+targets+of+DAF-16/FoxO.">DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO.</a> Mol Syst Biol. 6, 399 (2010).</li><li>Bianchi-Frias, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hairy+transcriptional+repression+targets+and+cofactor+recruitment+in+Drosophila.">Hairy transcriptional repression targets and cofactor recruitment in Drosophila.</a> PLoS Biol. 2, e178 (2004).</li><li>Woolcock, K. J., Gaidatzis, D., Punga, T., Buhler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dicer+associates+with+chromatin+to+repress+genome+activity+in+Schizosaccharomyces+pombe.">Dicer associates with chromatin to repress genome activity in Schizosaccharomyces pombe.</a> Nat Struct Mol Biol. 18, 94-99 (2011).</li><li>Luo, S. D., Shi, G. W., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+targets+of+the+D.+melanogaster+DSXF+protein+and+the+evolution+of+sexual+development.">Direct targets of the D. melanogaster DSXF protein and the evolution of sexual development.</a> Development. 138, 2761-2771 (2011).</li><li>Germann, S., Gaudin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+in+vivo+protein-DNA+interactions+in+plants+by+DamID,+a+DNA+adenine+methylation-based+method.">Mapping in vivo protein-DNA interactions in plants by DamID, a DNA adenine methylation-based method.</a> Methods Mol Biol. 754, 307-321 (2011).</li><li>Zhang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+LHP1+protein+colocalizes+with+histone+H3+Lys27+trimethylation.">The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation.</a> Nat Struct Mol Biol. 14, 869-871 (2007).</li><li>Orian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+profiling,+DamID+and+the+emerging+landscape+of+gene+expression.">Chromatin profiling, DamID and the emerging landscape of gene expression.</a> Curr Opin Genet Dev. 16, 157-164 (2006).</li><li>Vogel, M. J., Peric-Hupkes, D., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+in+vivo+protein-DNA+interactions+using+DamID+in+mammalian+cells.">Detection of in vivo protein-DNA interactions using DamID in mammalian cells.</a> Nat Protoc. 2, 1467-1478 (2007).</li><li>Greil, F., Moorman, C., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DamID:+mapping+of+in+vivo+protein-genome+interactions+using+tethered+DNA+adenine+methyltransferase.">DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase.</a> Methods Enzymol. 410, 342-359 (2006).</li><li>de Wit, E., Greil, F., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+HP1+binding+in+Drosophila:+developmental+plasticity+and+genomic+targeting+signals.">Genome-wide HP1 binding in Drosophila: developmental plasticity and genomic targeting signals.</a> Genome Res. 15, 1265-1273 (2005).</li><li>. Illumina TruSeq adaptors &amp; PCR primers Available from: <a target="_blank" href="https://ethanomics.wordpress.com/chip-seq-library-construction-using-the-illumina-truseq-adapters/">https://ethanomics.wordpress.com/chip-seq-library-construction-using-the-illumina-truseq-adapters/</a> (2015)</li><li>. Optimization of PCR cycles for NGS Available from: <a target="_blank" href="https://ethanomics.wordpress.com/ngs-pcr-cycle-quantitation-protocol/">https://ethanomics.wordpress.com/ngs-pcr-cycle-quantitation-protocol/</a> (2015)</li><li>Bernstein, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NIH+Roadmap+Epigenomics+Mapping+Consortium.">The NIH Roadmap Epigenomics Mapping Consortium.</a> Nat Biotechnol. 28, 1045-1048 (2010).</li><li>Encode Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+user's+guide+to+the+encyclopedia+of+DNA+elements+(ENCODE).">A user's guide to the encyclopedia of DNA elements (ENCODE).</a> PLoS Biol. 9, e1001046 (2011).</li><li>Asp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+remodeling+of+the+epigenetic+landscape+during+myogenic+differentiation.">Genome-wide remodeling of the epigenetic landscape during myogenic differentiation.</a> Proc Natl Acad Sci U S A. 108, E149-E158 (2011).</li><li>Hoppe, P. S., Coutu, D. L., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+technologies+sharpen+up+mammalian+stem+cell+research.">Single-cell technologies sharpen up mammalian stem cell research.</a> Nat Cell Biol. 16, 919-927 (2014).</li><li>Avital, G., Hashimshony, T., Yanai, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing:+new+methods+for+in+situ+single-cell+transcriptomics.">Seeing is believing: new methods for in situ single-cell transcriptomics.</a> Genome Biol. 15, 110 (2014).</li><li>Navin, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+genomics:+one+cell+at+a+time.">Cancer genomics: one cell at a time.</a> Genome Biol. 15, 452 (2014).</li><li>Saliba, A. E., Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-seq:+advances+and+future+challenges.">Single-cell RNA-seq: advances and future challenges.</a> Nucleic Acids Res. 42, 8845-8860 (2014).</li><li>Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+Hi-C+reveals+cell-to-cell+variability+in+chromosome+structure.">Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.</a> Nature. 502, 59-64 (2013).</li><li>Kind, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+dynamics+of+genome-nuclear+lamina+interactions.">Single-cell dynamics of genome-nuclear lamina interactions.</a> Cell. 153, 178-192 (2013).</li><li>Southall, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+profiling+of+gene+expression+and+chromatin+binding+without+cell+isolation:+assaying+RNA+Pol+II+occupancy+in+neural+stem+cells.">Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells.</a> Dev Cell. 26, 101-112 (2013).</li></ol>

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 ...

DNA adenin metiltransferaz tanımlaması (DamID) 1,2 in vivo protein-DNA etkileşimlerini tespit etmek için bir yöntem olup, kromatin, immüno-çökeltme (chip) 3 için alternatif bir yöntemdir. Hücrelerin nispeten düşük bir miktarda kullanılır ve DNA ya da yüksek ölçüde spesifik bir antikor, proteinin kimyasal olarak çapraz-bağlanmasını gerektirmez. Hedef protein gevşek veya dolaylı olarak DNA ile ilişkili olduğunda ikincisi özellikle yararlıdır. DamID başarılı bir şekilde çekirdek zarı proteinlerinin 4-10, kromatin ilişkili proteinler 11-13, kromatin değiştirici enzimler 14, transkripsiyon faktörleri ve 15-18 ko-faktörler ve RNAi makine 19 de dahil olmak üzere bir çok protein bağlanma sitesi eşlemek için kullanılmıştır. Yöntem S. dahil olmak üzere birden organizmalarda uygulanabilir cerevisiae 13, S. pombe 7, C. elegans 9,17, D. melanogaster 5,11,18,20, A. 21,22 yanı sıra, fare ve insan hücre hatları 6,8,10,23,24 thaliana. DamID deneyinin geliştirilmesi endojen adenin metilasyonu 2 eksikliği ökaryotik hücrelerde adenin-metillenmiş DNA fragmanlarının spesifik olarak saptanması dayanıyordu. Ilgi ve E, DNA bağlama proteini aşağıdakilerden oluşan bir füzyon proteini, coli DNA adenin metiltransferaz (Baraj), mekansal yakınlık (en önemlisi 1 kb içinde ve yukarı yaklaşık 5 kb) olan genom 2 protein bağlanma sitelerine GAİK dizileri adenin baz metillenmesi olabilir. Tadil edilmiş DNA fragmanları, özellikle ilgi 1,25,26 proteinin genomik bağlanma bölgelerini tespit etmek için microarrays yükseltilir ve hibridize edilebilir. Bu orijinal DamID yöntemi mikrodizilerinin kullanılabilirliği ve önceden belirlenmiş prob yoğunluğu ile sınırlı kalmıştır. Bu nedenle, yüksek verimli sekanslamayı entegre etmişDamID 10 ve DamID-SEQ olarak yöntem belirlenmiş. Kısacası çok sayıda DamID-seq oluşturulan okur genom protein-DNA etkileşimleri hassas yerelleştirme sağlar. Biz DamID seq genom nükleer lamina (NL) dernekleri 10 çalışmak için mikrodizi tarafından DamID daha yüksek çözünürlük ve daha geniş bir dinamik aralık sağlanan bulundu. Bu geliştirilmiş bir yöntem gen yapıları 10 içinde NL dernek sondalama izin verir ve bu ChIP-seq ve RNA-seq gibi diğer yüksek verim sıralama verileri ile karşılaştırmalar kolaylaştırır. Burada anlatılan DamID seq protokol başlangıçta haritalama genom NL dernekler 10 için geliştirilmiştir. Bu E. fare ya da insan Lamin B1 hayvan zinciri ile bir füzyon proteini oluşturulur coli DNA adenin metiltransferaz ve C2C12 fare (veri yayınlanmadı) 10 ve IMR90 insan fetal akciğer fibroblastlar miyoblastların, 3T3 fare embriyonik fibroblast protokolü test. Bu protokol, biz c ile başlayanvektörleri onstructing ve memeli hücrelerinde 24 lentiviral enfeksiyon Barajı-gergin füzyon proteinleri ifade. Sonra, adenin-metillenmiş DNA fragmanları amplifiye ve diğer organizmalarda uygulanabilir olmalı dizileme kitaplıkları hazırlama ayrıntılı protokolleri açıklar.

<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.

Baraj V5-LmnB1 füzyon proteini, immünofloresan boyama endojen lamin B proteini (Şekil 1) ile birlikte lokalize olduğu teyid edildi. Adenin-metillenmiş DNA fragmanlarının PCR amplifikasyonu, başarılı DamID-SEQ için önemli bir adımdır. No-veya açıkça daha az büyütme (Şekil 2) yol açmalıdır (ligaz olmadan ya da PCR şablon olmadan, DpnI olmadan) negatif kontrol ederken 2 kb...

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

Biz yüksek verimlilik sıralama (DamID seq) DNA adenin metiltransferaz kimlik (DamID) bağlanması burada bir tahlil açıklar. Bu geliştirilmiş bir yöntem daha yüksek çözünürlüğü ve daha geniş bir dinamik aralığı ve karşılaştırın vb ChIP-SEQ RNA-SEQ gibi diğer yüksek verimli sekanslama verileri ile birlikte DamID seq verileri analiz

DamID seq: Adenin-metillenmiş DNA Fragments Yüksek Verimli Dizileme tarafından Protein-DNA Etkileşimleri genom Haritalama

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

Research

JoVE Journal

Biology

Electroeluting DNA Fragments

DNA Fragments Electroeluting

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

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

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

Nicel ve Otomatik Yüksek throughput Genom çapında RNAi Ekranlar 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. ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Otomatik kullanarak Kromozomlar Yüksek throughput Fiziksel Haritalama<em&gt; In situ</em&gt; Hibridizasyon

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

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

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

Genom çapında Gen Delesyonlar içinde<em&gt; Streptococcus sanguinis</em&gt; Yüksek Verimli PCR ile

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)

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

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (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

Protein-DNA Etkileşiminin Çalışmaları Tek molekül Manipülasyon ve Görüntüleme birleştiren

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

Yüksek Verim Dizilimine tarafından MicroRNA ölçümü için Küçük RNA molekülleri, yüksek Verimli ligasyonu

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

DNA Metilasyon Analizi nesil Dizilimine tarafından hedef

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

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

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

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

Arabidopsis Protein-DNA Etkileşimleri Tespiti için Kromatin Immunoprecipitation Deneyi<em&gt; İn Vivo</em

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