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>
	<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>

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 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 &amp;amp; Tissue Kit (250)</td><td>QIAGEN</td><td>69506 or 69504&amp;nbsp;&amp;nbsp;</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>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/25300054&quot;&gt;Trypsin-EDTA (0.05%), phenol red&lt;/a&gt;</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 &quot;AdRt&quot; and &quot;AdRb&quot;</td><td>Integrated DNA Technologies</td><td></td><td>sequences available in ref. 24; no phosphorylation of the 5&#039; or 3&#039; 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 &quot;AdR_PCR&quot;</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&amp;nbsp; 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 &amp;micro;g/&amp;micro;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 &amp;micro;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 &amp;micro;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&amp;rsquo; &amp;#8594; 5&amp;rsquo; 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 3.4.3; 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 10 mM 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 &amp;#956;m 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 - deney örnekleri 0.2 bir karalama yükseltmek gerekir. Bir NGS kütüphane için arzu edilen uç boyutu 200 ila 300 bp ise metillenmiş DNA fragmanları, 0.2 2 kb arasında aralığındadır. Bu nedenle, uygun bir boyut aralığına metil PCR ürünleri parçalara esastır. Bununla birlikte, aynı anda bir uygun boyutta aşağı daha geniş DNA fragmanlarını kırmak için pratik olduğu bulunmuşturs ve tek bir parçalanma süresi sağlam küçük DNA parçalarının çoğunluğu tutun. Bu nedenle, zaman süreçli deneyler, 200 bp (Şekil 3) merkezlenen bir lekelenmesine parçası 1 ug DNA için gereken minimum süre (T 0.2kb) belirlemek için yapıldı. Sonra eşit artışlarla 6 sürelerde gerçek parçalanma 5 dakika ile T 0.2kb arasında seçildi. Çift iplikli DNA&#39;nın Fragmentase enzimatik aktivitesi, partiden partiye değişebilir ve zamanla azalabilir, bu nedenle Fragmentase yeni bir parti ya da belirli bir süre depolandıktan sonra bu adımı tekrar tavsiye edilir. İstenen uç boyutlu agaroz jeli üzerinde 300 ve (121 bp dizi adaptörleri de dahil olmak üzere), 400 bp arasında, DNA fragmanlarına karşılık gelen 200 ve 300 bp. Bu aralıkta üç ince dilim bir kütüphane boyutu aralığını daraltmak ve olasılığını artırmak için her deney örnek kesilmiştirEn az bir nitelikli sıralama kitaplığı (Şekil 4) elde. Her bir amplifiye DNA kütüphanesinin 5 ul&#39;lik bir tümböleni sekanslama için uygun olabilir, ki kütüphane belirlemek için agaroz jeli üzerinde analiz edilmiştir. Şekil 5A&#39;da gösterildiği gibi, kesilmiş jel dilimine aynı boyutta açık bir tek bant agaroz jel (aşama 3.7.4) görünür olmalıdır. Daha sonra, seçilen kütüphaneleri dizileme önce kesin boyut aralığı ve konsantrasyonlarını belirlemek için bir Bioanalyzer (Şekil 5B) ile incelenmiştir. Arzu edildiği takdirde, amplifiye DNA kütüphaneleri ile doğrudan jel analizi olmaksızın Bioanalyzer ile incelenebilir. Birden kütüphaneler kaliteli olduğunda, benzer büyüklükteki kütüphaneleri örnekleri (V5-Baraj ifade eden hücreler) deneysel (Baraj-V5-POI salgılayan hücrelerin) ve kontrol bir çift aralıkları sıralamak için tavsiye edilir. Kısacası tarafından oluşturulan okursıralama sistemleri ilk olarak geri gelen genom haritası çizilmiştir bulundu. Benzersiz daha sonraki analizlere geçirildi okur hizalanmış. Kısa işlemek için bir boru hattı, bir genom-NL etkileşim haritası oluşturmak ve gen-NL dernekler önceki çalışmaları 10 ayrıntılı olarak tarif edilmiştir analiz okur. Örnek sonuçlar Şekil 6&#39;da gösterilmektedir. <img alt="figür 1" src="/files/ftp_upload/53620/53620fig1.jpg" /> Immünofloresan boyama ile füzyon proteinlerinin çekirdek içi lokalizasyonu doğrulama Şekil 1.. IMR90, insan akciğer fibroblast hücreleri, geçici olarak Dam-V5-LmnB1 eksprese eden bir plazmid ile transfekte edildi ve anti-lamin B (A, kırmızı) ve anti-V5 (B ile boyandı yeşil). (C) A ve B görüntüleri birleştirme <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig1large.jpg" target="_blank">t büyük halini görmek için tıklayınız</a>Onun rakam. <img alt="Şekil 2," src="/files/ftp_upload/53620/53620fig2.jpg" /> Adenin-metillenmiş DNA fragmanları amplifiye Şekil 2. PCR. Her bir PCR reaksiyonundan kaynaklanan, 5 ul bir alikosu, bir% 1 agaroz jeli üzerinde analiz edilmiştir. 0.2 2 kb arasında değişen bir smear her deney numuneden amplifiye edildi, ancak hiçbir amplifikasyon negatif kontroller gözlendi (adım 2.1 hiçbir DpnI, adım 2.2 veya hiçbir PCR şablonunda hiçbir Ligaz). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig2large.jpg" target="_blank">Tıklayınız daha büyük bir versiyonunu görmek için bu rakamın.</a> <img alt="Şekil 3," src="/files/ftp_upload/53620/53620fig3.jpg" /> Şekil 3. Optimal parçalanma süreleri belirlenmesi. Arıtılmış metil PCR ürünleri 10 dakikalık bir artışında 5 ila 55 dakika zaman süreleri için parçalanma tabi tutulmuştur (sindirilmemiş DNA<html> &quot;0 dak&quot;) olarak etiketlenmiş. 1 DNA merdiveni ug ve her bir parçalanmış DNA örneği 0.5 ug bir agaroz jeli üzerinde analiz edilmiştir. Yaklaşık 200 bp DNA smear çoğunluğu sindirmek için çok az zaman 5 ile 35 dakika (5 ve 35 dk dahil) arasında altı eşit aralıklı sürelerde gerçek parçalanma gerçekleştirmek için seçildi nedenle yaklaşık 35 dakika, olduğu belirlendi. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig3large.jpg" target="_blank">Lütfen Bu rakamın büyük halini görmek için buraya tıklayın.</a> <img alt="Şekil 4," src="/files/ftp_upload/53620/53620fig4.jpg" /> Şekil 4. Boyut adımı 3.6% 2 agaroz jel üzerinde yürütülmüştür dan. Baraj-V5-LmnB1 ve V5-Baraj DNA örnekleri DNA kütüphanelerinin seçme ve üç eşit büyüklükteki jel dilimleri (L, M ve H düşüğe gelen orta sarı çizgilerle gösterildiği gibi) boyutu yüksek 300 ve 400 bp arasında çıkarılmıştır.<html> om / files / ftp_upload / 53620 / 53620fig4large.jpg &quot;target =&quot; _ blank &quot;&gt; bu rakamın büyük halini görmek için lütfen buraya tıklayınız. <img alt="Şekil 5," src="/files/ftp_upload/53620/53620fig5.jpg" /> Bir agaroz jeli üzerinde analiz Şekil yüksek sekanslama 5. Kuvvetlendirilmiş DNA kütüphaneleri (A). Amplifiye DNA kütüphaneleri. PCR şablonları Şekil 4 üzerinde gösterilen jel dilimlerinden saflaştırılmıştır. (B) V5-Baraj (L) numune ve (A) &#39;de vurgulanmıştır Dam-V5-LmnB1 (L) numunenin Bioanalyzer sonuçlanır. Bu iki kütüphaneler benzer dar boyut aralıkları vardı ve yüksek verimlilik sıralaması dolayısıyla nitelikli. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig5large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> jpg &quot;/&gt; UCSC genom tarayıcısı. Fare kromozom 1 görüntülenen Şekil 6. NGS veri örnek olarak gösterilmektedir. Dizi verileri, fare C2C12 miyoblastların 10 elde edildi. Sırasıyla Barajı-V5-LmnB1 ve V5-Barajı&#39;nı salgılayan hücrelerin verilerine karşılık gelen İzleri &quot;MB.LmnB1.w2k&quot; ve &quot;MB.Dam.w2k&quot;, arsa normalize okuma yoğunlukları (milyon benzersiz okur, ya da RPKM eşlenen başına kilobaz başına okur ) kromozom boyunca örtüşmeyen ardışık 2 kb pencerelerde. Parça &quot;MB.log2FC.w2k&quot; araziler genom NL dernekler, yani log2 2 kb çözünürlükte Barajı üzerinde LmnB1 ve RPKM oranları. Sıralama tabanlı Lamina İlişkili Alanlar (sLADs, istatistiksel öneme sahip Barajı üzerinde LmnB1 yüksek okuma yoğunlukları var yani genomik bölgeleri) beyaz, gri ve belirsiz bölgelerde siyah, sigara sLADs boyar &quot;MB.sLADs&quot; izleyin. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53620/53620fig6large.jpg" target="_blank">Için tıklayınız Yazılımı yükle</a>Bu dosyayı d.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biology

19.3K Görüntüleme.  Yale University School of Medicine.  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 

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

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Hasta Dokularda metil-bağlayıcı DNA yakalama Dizi

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Genetik

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

ChEC-seq ile Protein-DNA Etkileşimlerinin Genom Boyunca Haritalandırılması<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

Hibridizasyon uyarlaması yakalama kromatin ilişkili proteinlerin proteomik memeli hücreleri için

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo) Kullanılarak Fare Kök Hücre Türevi Nöronlarda Protein-DNA Etkileşimlerinin Yüksek Çözünürlüklü Haritalanması

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

Mapping Mammalian 3D Genome Interactions with Micro-C-XL

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, known as enhancers, are thought to regulate the activity of distal promoters via interaction in 3D space. Genome-wide chromosome conformation capture (3C)-technologies, such as Hi-C, have transformed our understanding of how genomes are organized in cells. The current understanding of 3D genome organization is limited by the resolution with which the topological organization of chromosomes in 3D space can be resolved. Micro-C-XL measures chromosome folding with resolution at the level of the nucleosome, the basic unit of chromatin, by utilizing micrococcal nuclease (MNase) to fragment genomes during the chromosome conformation capture protocol. This results in an improved signal-to-noise ratio in the measurements, thus facilitating the better detection of insulation sites and chromosome loops compared to other genome-wide 3D technologies. A visually supported, detailed, step-by-step protocol for preparing high-quality Micro-C-XL samples from mammalian cells is presented in this article.

A protocol for mapping the three-dimensional genome organization with nucleosome resolution using the genome-wide chromosome conformation capture method Micro-C-XL is presented here.

Memeli 3D Genom Etkileşimlerinin Micro-C-XL ile Haritalanması

Three-dimensional (3D) chromosome organization is a major factor in genome regulation and cell-type specification. For example, cis-regulatory elements, ...

<meta charset="utf8"></head><body>CUT&RUN veri analizi için yeni başlayanlar için uygun tek dilli ardışık düzen

Introductory Analysis and Validation of CUT&#38;RUN Sequencing Data

The CUT&amp;RUN technique facilitates detection of protein-DNA interactions across the genome. Typical applications of CUT&amp;RUN include profiling changes in histone tail modifications or mapping transcription factor chromatin occupancy. Widespread adoption of CUT&amp;RUN is driven, in part, by technical advantages over conventional ChIP-seq that include lower cell input requirements, lower sequencing depth requirements, and increased sensitivity with reduced background signal due to a lack of cross-linking agents that otherwise mask antibody epitopes. Widespread adoption of CUT&amp;RUN has also been achieved through the generous sharing of reagents by the Henikoff lab and the development of commercial kits to accelerate adoption for beginners. As technical adoption of CUT&amp;RUN increases, CUT&amp;RUN sequencing analysis and validation become critical bottlenecks that must be surmounted to enable complete adoption by predominantly wet lab teams. CUT&amp;RUN analysis typically begins with quality control checks on raw sequencing reads to assess sequencing depth, read quality, and potential biases. Reads are then aligned to a reference genome sequence assembly, and several bioinformatics tools are subsequently employed to annotate genomic regions of protein enrichment, confirm data interpretability, and draw biological conclusions. Although multiple in silico analysis pipelines have been developed to support CUT&amp;RUN data analysis, their complex multi-module structure and usage of multiple programming languages render the platforms difficult for bioinformatics beginners who may lack familiarity with multiple programming languages but wish to understand the CUT&amp;RUN analysis procedure and customize their analysis pipelines. Here, we provide a single-language step-by-step CUT&amp;RUN analysis pipeline protocol designed for users with any level of bioinformatics experience. This protocol includes completing critical quality checks to validate that the sequencing data is suitable for biological interpretation. We expect that following the introductory protocol provided in this article combined with downstream peak annotation will allow users to draw biological insights from their own CUT&amp;RUN datasets.

<meta charset="utf8"></head><body>Yazar Gündemi: Kanser Başlangıcı ve İmmünoterapi Direncinde Tekrarlayan DNA Yanlış Düzenlemesinin Rolünün Araştırılması

This protocol guides bioinformatics beginners through an introductory CUT&amp;RUN analysis pipeline that enables users to complete an initial analysis and validation of CUT&amp;RUN sequencing data. Completing the analysis steps described here, combined with downstream peak annotation, will allow users to draw mechanistic insights into chromatin regulation.

CUT&#38;RUN Sıralama Verilerinin Giriş Analizi ve Doğrulanması

The CUT&amp;RUN technique facilitates detection of protein-DNA interactions across the genome. Typical applications of CUT&amp;RUN include profiling ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

DNA-Protein Etkileşimlerini İncelemek için CD Spektroskopisi

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Biyokimya

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

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

Özet

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Hasta Dokularda metil-bağlayıcı DNA yakalama Dizi

ChEC-seq ile Protein-DNA Etkileşimlerinin Genom Boyunca Haritalandırılması

Hibridizasyon uyarlaması yakalama kromatin ilişkili proteinlerin proteomik memeli hücreleri için

Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo) Kullanılarak Fare Kök Hücre Türevi Nöronlarda Protein-DNA Etkileşimlerinin Yüksek Çözünürlüklü Haritalanması

Memeli 3D Genom Etkileşimlerinin Micro-C-XL ile Haritalanması

CUT&RUN Sıralama Verilerinin Giriş Analizi ve Doğrulanması

CUT&RUN Sıralama Verilerinin Giriş Analizi ve Doğrulanması

CUT&RUN Sıralama Verilerinin Giriş Analizi ve Doğrulanması

DNA-Protein Etkileşimlerini İncelemek için CD Spektroskopisi

Kromatin Etkileşimler haritalama ve Transkripsiyon Yönetmelik anlamak için paired-end Tag Dizileme (Chia-PET) ile Kromatin Etkileşim Analizi