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.产生和融合蛋白和自由大坝蛋白的表达<ol><li> 感兴趣进入DamID载体克隆蛋白。 <ol><li>根据制造商的方案扩增使用所需的高保真DNA聚合酶的兴趣点（POI）的蛋白质和合适的引物的cDNA的。实验确定最佳扩增条件，以确保插入适当的放大。 </li><li>运行一个琼脂糖凝胶并根据制造商的方案纯化的POI扩增的cDNA由凝胶提取试剂盒。 </li><li>克隆的cDNA的POI成采用BP克隆酶II根据制造商的协议的pDON...

<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 ...

脱氧核糖核酸腺嘌呤甲基识别（DamID）1,2是这样一种方法来检测体内蛋白-DNA相互作用，是一种可供选择的方法来染色质免疫沉淀（ChIP）3。它采用的是相对低的细胞的量，并且不需要化学交联蛋白与DNA或高度特异性的抗体。后者是特别有用当靶蛋白是松散或间接地与DNA相关联。 DamID已成功地用于绘制的多种蛋白质，包括核包膜蛋白4-10，相关联的染色质蛋白11-13染色质修饰酶14，转录因子和辅因子15-18和RNAi机械19的结合位点。该方法适用于多种生物，包括S.酵母 13，S。酵母 7，C。线虫 9,17，D。黑 5,11,18，20，A。拟南芥 21,22以及小鼠和人的细胞系6,8,10,23,24。 所述DamID测定的发展是基于在缺乏内源性腺嘌呤甲基化2的真核细胞腺嘌呤甲基化的DNA片段的特异性检测。表达的融合蛋白，包括利息和 E的DNA结合蛋白的大肠杆菌DNA腺嘌呤甲基（水坝），可以甲基化腺嘌呤碱中GATC序列是在空间接近性（最显著内1 kb和多达大约5kb的）的蛋白质的基因组中的2的结合位点。修饰的DNA片段可以特异性地扩增和杂交到微阵列检测感兴趣1,25,26的蛋白质的基因组结合位点。这个原始DamID方法是由微阵列的可用性和预定探针的密度的限制。因此，我们集成了高通量测序到DamID 10和指定的方法DamID-起。读取从DamID-SEQ产生大量的短使蛋白质-DNA相互作用的全基因组的精确定位。我们发现，DamID-SEQ提供比DamID更高的分辨率和更宽的动态范围芯片用于研究基因组核纤层（NL）协会10。这种改进的方法允许基因结构 10内探测NL协会和便于与其它高通量测序数据，如芯片起和RNA-SEQ比较。 这里描述的DamID-SEQ协议最初开发用于映射基因组-NL协会10。我们通过圈养小鼠或人类核纤层蛋白B1至E.产生的融合蛋白大肠杆菌DNA甲基腺嘌呤和测试协议中3T3小鼠胚胎成纤维细胞，C2C12小鼠成肌细胞10和IMR90人胚肺成纤维细胞（数据未公布）。在这个协议中，我们开始用Constructing载体和表达坝-拴系融合蛋白由慢病毒感染的哺乳动物细胞24。接着，我们描述放大腺嘌呤甲基化的DNA片段，并准备测序文库，应该适用于其它生物体的详细方案。

<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.

大坝-V5-LmnB1融合蛋白被确认已共定位与内源拉明乙蛋白通过免疫荧光染色（ 图1）。 腺嘌呤甲基化DNA片段的成功的PCR扩增为DamID-SEQ的关键步骤。实验样品应扩增的0.2污迹- 2 kb的，而阴性对照（无DPNI，没有连接酶或不含PCR模板）应导致无或明显较少的扩增（ 图2）。 <p class="jove_content" fo:keep-together.with...

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

我们描述这里的一个检测通过结合DNA腺嘌呤甲基识别（DamID），以高通量测序（DamID-SEQ）。这种改进的方法提供了更高的分辨率和更宽的动态范围，并允许在分析与其他高通量测序数据如芯片起，RNA测序等一起DamID-SEQ数据

DamID-SEQ：蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

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

Electroeluting的DNA碎片

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

科研

生物学

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

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

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</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

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

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

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

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

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

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

结合单分子操纵和成像蛋白-DNA相互作用的研究

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

小RNA分子的高效结扎的微小RNA定量高通量测序

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

有针对性的DNA甲基化分析通过新一代测序

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

高通量筛选转录后调控基因的化学调节剂

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

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

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