Os autores são apoiados por A * STAR de Cingapura. Além disso, MJF é apoiada por um Nacional ESTRELA A * Bolsa de Ciência, L&#39;Oréal para Mulheres na Ciência Nacional Fellowship e Lee Kuan Yew Sociedade Pós-Doutorado. YR é suportado pelo NIH (R01 Codificar subsídios HG004456-01 e R01 HG003521-01). Os autores também reconhecem a equipe de vídeo de 8 Productions Pixels, de Cingapura, em especial o Sr. Kelvin Issey, Chang Kai Xiang Sr. e Sherwin Mr. Gan para filmar as cenas, a Sra. Siti Rahim para edição de vídeo e Michelle Sra. Teo para o voice-over.

<ol>
	<li>Cai, S., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SATB1+packages+densely+looped,+transcriptionally+active+chromatin+for+coordinated+expression+of+cytokine+genes.">SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.</a> Nat. Genet. 38, 1278-1288 (2006).</li><li>Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+replication+and+transcription.">The organization of replication and transcription.</a> Science. 284, 1790-1795 (1999).</li><li>Das, P. M., Ramachandran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+assay.">Chromatin immunoprecipitation assay.</a> Biotechniques. 37, 961-969 (2004).</li><li>Deng, W., Blobel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+chromatin+loops+provide+epigenetic+gene+expression+states.">Do chromatin loops provide epigenetic gene expression states.</a> Curr. Opin. Genet. Dev. 20, 548-554 (2010).</li><li>Fullwood, M. J., Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+interaction+analysis+using+paired-end+tag+sequencing.">Chromatin interaction analysis using paired-end tag sequencing.</a> Curr. Protoc. Mol. Biol. Chapter 21, 21-25 (2010).</li><li>Fullwood, M. J., Liu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+oestrogen-receptor-alpha-bound+human+chromatin+interactome.">An oestrogen-receptor-alpha-bound human chromatin interactome.</a> Nature. 462, 58-64 (2009).</li><li>Jackson, D. A., Hassan, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+focal+sites+of+transcription+within+human+nuclei.">Visualization of focal sites of transcription within human nuclei.</a> EMBO J. 12, 1059-1065 (1993).</li><li>Li, G., Fullwood, 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=ChIA-PET+tool+for+comprehensive+chromatin+interaction+analysis+with+paired-end+tag+sequencing.">ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing.</a> Genome Biol. 11, R22 (2010).</li><li>Li, G., Ruan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Promoter-Centered+Chromatin+Interactions+Provide+a+Topological+Basis+for+Transcription+Regulation.">Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation.</a> Cell. 148, 84-98 (2012).</li><li>Ng, P., Wei, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired-end+diTagging+for+transcriptome+and+genome+analysis.">Paired-end diTagging for transcriptome and genome analysis.</a> Curr. Protoc. Mol. Biol. Chapter. Chapter 21, 12 (2007).</li><li>Nowak, D. E., Tian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+cross-linking+method+for+identification+of+NF-kappaB+gene+network+by+chromatin+immunoprecipitation.">Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.</a> Biotechniques. 39, 715-725 (2005).</li><li>Osborne, C. S., Chakalova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+genes+dynamically+colocalize+to+shared+sites+of+ongoing+transcription.">Active genes dynamically colocalize to shared sites of ongoing transcription.</a> Nat. Genet. 36, 1065-1071 (2004).</li><li>Rippe, K., von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+at+a+distance:+DNA-looping+and+initiation+of+transcription.">Action at a distance: DNA-looping and initiation of transcription.</a> Trends Biochem. Sci. 20, 500-506 (1995).</li><li>Schoenfelder, S., Sexton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+associations+between+co-regulated+genes+reveal+a+transcriptional+interactome+in+erythroid+cells.">Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.</a> Nat. Genet. 42, 53-61 (2010).</li><li>Zeng, P. Y., Vakoc, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+dual+cross-linking+for+identification+of+indirect+DNA-associated+proteins+by+chromatin+immunoprecipitation.">In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation.</a> Biotechniques. 41, 694-698 (2006).</li></ol>

Não há conflitos de interesse declarados.

Chia-PET é um método desenvolvido para identificar interações de longo alcance na regulação da transcrição. Um dos factores críticos que determinam a qualidade de uma biblioteca de Chia-PET é a qualidade do material ChIP. O protocolo mostrado no vídeo incorpora o uso de EGS e formaldeído para reticular as células. A utilização de formaldeído combinado com um reagente de ligação cruzada tendo um segundo braço mais longo espaçador pode ajudar na ligação de proteínas que não podiam ser ligados por formaldeído sozinho 3,11,15. Foram construídas bibliotecas com este método que demonstraram robustos sítios de ligação e de longo alcance das interacções 9. No entanto, a reticulação e das condições de chip deve ser optimizada para cada factor de interesse, e é importante para não sobre-reticulação como demasiada reticulação irá resultar em dificuldades de fragmentação por sonicação, e pode possivelmente resultar em interacções de cromatina espúrios . Interações de cromatinaidentificado por Chia-PET deve ser validado por um método diferente, tais como hibridação in-situ de fluorescência 4. Recomendamos um mínimo de 100 ng de material de cromatina. Embora tenhamos construído bibliotecas de boa qualidade a partir de 50 ng de material de cromatina, observou-se que grandes quantidades de material de partida permitiu a construção de bibliotecas de Chia-PET com menos de 16 ciclos de PCR, amplicons, minimizando assim e redundância de cada biblioteca. Esta menor redundância correlacionada com a maior únicas marcas mapeadas e também uma alta porcentagem de dados úteis, permitindo assim um mapa de interação mais abrangente cromatina com pistas menos de seqüenciamento. O volume final embalado de grânulos em cada tubo deve ser de 50 uL e 100 uL de pérolas magnéticas e Sepharose respectivamente. Se o volume embalado grânulo é menos do que o indicado, para trazer o volume mínimo embalado com similarmente pré-limpos em branco esferas magnéticas ou Sepharose para minimizar a perda de DNA-rolamento de esferass nas etapas subseqüentes. Cano serrado dicas ou grande-core dicas devem ser usadas para pipetagem contas Sepharose. As modificações que se seguem foram incorporados na sequência do publicado anteriormente Chia-PET protocolo 5. Em primeiro lugar, as esferas magnéticas g foram utilizados para minimizar a perda de amostra durante as lavagens. Além disso, identificou-se as bandas não específicas com tamanhos aproximados de 100 pb e 138 pb para ser amplicões de auto-ligadura meias-linkers e / ou adaptadores. Assim, nós reduziu a concentração de biotinilados meias-linkers e 454 GS20 adaptadores para minimizar as bandas não específicas durante a amplificação por PCR. O volume ligadura proximidade foi reduzida de 50 ml a 10 ml para minimizar a perda de amostra durante os passos de purificação subsequentes e também economizar nos custos de reagentes. Nós também aumentou o tempo de incubação para imobilizar Chia-PET DNA para grânulos para garantir a captura máxima de Chia-PET ADN sobre as pérolas de estreptavidina. Durante o passo de ligação proximidade, ligaduras quiméricos that não representam verdadeira de interacções in vivo de cromatina são inevitavelmente gerado de uma forma não-específica e aleatórios. Assim, para avaliar a qualidade dos dados a partir de qualquer experiência Chia-PET, a taxa de quimerismo é estimado a partir da utilização de dois diferentes meias-linkers com específica de nucleótidos códigos de barras e TAAG ATGT 5. Depois de alto rendimento sequenciação, as sequências de Chia-PET são primeiro analisado para uma composição de código de barras linker e sequências derivadas de produtos de ligação específicos e produtos não específicos de ligação podem ser distinguidos 8. A percentagem de quimeras conhecidos (isto é, ligantes heterodímeros AB) apresentam no nosso em casa MCF-7 a RNA polimerase II Chia-PET bibliotecas é menos do que 15%. Chia-PET sequências são posteriormente classificadas em duas categorias, a saber, auto-ligadura os animais de estimação e laqueadura inter-PETS. Auto-ligação TAP são obtidos a partir de auto-circularização ligadura dos fragmentos de cromatina, enquanto a ligadura inter-TAP são derivados a partir de inter ligadura entre dois fragmentos de DNA diferentes. Este último é, então, sub-divididos em três categorias diferentes com base na distância genômico de cada tag no mesmo cromossomo (intracromossômica ligadura inter-PET) ou que ambas as marcas são mapeados para dois cromossomos diferentes (intercromossômicas ligadura inter-PET). Nós desenvolvemos um Chia-PET pacote de software ferramenta para classificar as diferentes categorias 8. Isto será baseado nos fragmentos de ADN que estão na biblioteca. Geralmente, os fragmentos menores chip vai dar uma maior resolução e cortado o para estes RNA polimerase II Chia-PET bibliotecas é de cerca de 4 kb. Além disso, as interacções de cromatina reais pode ser distinguida de ruído aleatório por contagem do número de ligadura inter-TAP em um cluster de interacção, por outras palavras, um aglomerado de contagem de PET de alta diz-se ter uma maior probabilidade de ser uma interacção real, 8 cromatina . Para filtrar os falsos positivos umsubir de âncoras altamente enriquecido, que pode formar a ligadura inter-PETs por acaso, um quadro de análise estatística também foi formulada para explicar a formação aleatória de algum animal de estimação de ligação inter-entre duas âncoras 8. Em conclusão, a técnica Chia-PET permite o mapeamento de redes de interação de cromatina em uma escala global. A implementação de ChIP em Chia-PET permite a redução da complexidade da biblioteca e ruído de fundo. Além disso, adiciona ChIP especificidade para as interacções de cromatina, permitindo o exame das interacções de cromatina específicos associados com factores de transcrição específicos 5.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de Catálogo </td><td> Comentários </td></tr><tr><td> 4-20% gradiente de TBE gel </td><td> Invitrogen </td><td> EC6225BOX </td><td> Passo B, 6,3 </td></tr><tr><td> 5 x tampão ligase T4 DNA com PEG </td><td> Invitrogen </td><td> 46300018 </td><td> Passo B, 2,2 </td></tr><tr><td> 6% de gel de TBE </td><td> Invitrogen </td><td> EC6263BOX </td><td> Passo B, 6,8 </td></tr><tr><td> Agilent DNA 100 Ensaio </td><td> Agilent Technologies </td><td> 5067-1504 </td><td rowspan="3"> Passo B, 7,1 </td></tr><tr><td> Agilent Ensaio DNA de alta sensibilidade </td><td> Agilent Technologies </td><td> 5067-4626 </td></tr><tr><td> Agilent Bioanalyzer 2100 </td><td> Agilent Technologies </td><td> G2940CA </td></tr><tr><td> Centrifugar Filtros Tubo (Spin-X) </td><td> Corning &lt;/ Td&gt; <td> CLS8160 </td><td> Passo B, 3,7 e 6,9 </td></tr><tr><td> Transilluminator Leitor escuro </td><td> Clare Chemical Research </td><td> DR46B </td><td> Passo B, 6,8 </td></tr><tr><td> Digital Celular Sonifier Disrupter </td><td> Branson </td><td> 450D-0101063591 </td><td> Passo A, 4,3 </td></tr><tr><td> DynaMag-2 ímã (Concentrador de Partículas Magnéticas) </td><td> Invitrogen </td><td> 123-21D </td><td> Passo A, Passo B 7.5.1, para todos os passos de lavagem esferas magnéticas </td></tr><tr><td> DynaMag-15 Ímã (Concentrador de Partículas Magnéticas) </td><td> Invitrogen </td><td> 123.01D </td><td> Passo A, 5 e 6 </td></tr><tr><td> DynaMag-PCR (Concentrador de Partículas Magnéticas) </td><td> Invitrogen </td><td> 49-2025 </td><td> Passo B, 6,5 </td></tr><tr><td> Escherichia coli DNA Polimerase I </td><td> NEB </td><td> M0209 </td><td> Passo B, 5,6 </td></tr><tr><td> GlycoBlue </td><td> Ambion </td><td> AM9516 </td&gt; <td> Passo A, Passo B 7.5.1, 4,3 e 6,5 </td></tr><tr><td> Illumina CBOT Sistema de Geração de Cluster </td><td> Illumina </td><td> SY-301-2002 </td><td> Passo B, 7,4 </td></tr><tr><td> Illumina Genome Analyzer IIx </td><td> Illumina </td><td> SY-301-1301 </td><td> Passo B, 7,5 </td></tr><tr><td> Illumina PE primers </td><td> Illumina </td><td> PE-102-1004 </td><td> Passo B, 5,4 (se necessário) </td></tr><tr><td> Intelli-Mixer </td><td> Palico Biotech </td><td> RM-2L </td><td> Passo B, quaisquer incubações com rotação </td></tr><tr><td> LightCycler 480 Real-Time PCR System </td><td> Roche </td><td> 04 640 268 001 </td><td> Passo A, Passo B 7.5.3, 7,3 </td></tr><tr><td> LightCycler480 DNA SYBR Green I MasterMix </td><td> Roche </td><td> 03 752 186 001 </td><td> Etapa B, 7.5.3 </td></tr><tr><td> M-280 Estreptavidina Dynabeads </td><td> Invitrogen </td><td> 11206D </td><td> Passo B, 5,2 </td></tr> &lt;tr&gt; <td> Magnética (Dynabeads) Proteína G </td><td> Invitrogen </td><td> 100.03D </td><td> Etapa A, 5.1.1 e 5.2.1 </td></tr><tr><td> MaXtract de Alta Densidade (2 ml) </td><td> Qiagen </td><td> 129056 </td><td> Passo A 7.5.1, </td></tr><tr><td> MaXtract de Alta Densidade (50 ml) </td><td> Qiagen </td><td> 129073 </td><td> Passo B, 4,2 </td></tr><tr><td> CEMm </td><td> NEB </td><td> R0637 </td><td> R0637 </td></tr><tr><td> RNase Ribonuclease ONE </td><td> Promega </td><td> M426C </td><td> Passo B, 4,8 </td></tr><tr><td> RNA polimerase II (8WG16) anticorpo monoclonal </td><td> Covance </td><td> MMS-126R </td><td> Passo A, 5.2.4 </td></tr><tr><td> Oak Ridge Tubos da centrífuga (polipropileno) </td><td> Nalgene </td><td> 3119-0050 </td><td> Passo A, 3,1 </td></tr><tr><td> Oak Ridge Tubos da centrífuga (Teflon FEP) </td><td> Nalgene </td><td> 3114-0050 </td><td> Passo B, 4,3 </td></tr><tr><td> Phusion Alta Fidelidade Master Mix </td><td> Finnzymes </td><td> F-531 </td><td> Passo B, 6 </td></tr><tr><td> Picogreen (Quant-iT) Reagente dsDNA </td><td> Invitrogen </td><td> P11495 </td><td> Passo A, 7.5.2 </td></tr><tr><td> Poliestireno Tubo de Ensaio de fundo redondo </td><td> BD Biosciences </td><td> 352057 </td><td> Passo A, 4,1 </td></tr><tr><td> Inibidor da Protease Comprimidos Cocktail (completo, EDTA-free) </td><td> Roche </td><td> 11873580001 </td><td> Etapa A, 1.6 em diante </td></tr><tr><td> Proteinase K Solução (20mg/ml) </td><td> Fermentas </td><td> E00491 </td><td> Passo A, 4,4, 7.5.1 Passo B, 4,1 </td></tr><tr><td> DNA-ligase de T4 </td><td> Fermentas </td><td> EL0013 </td><td> Passo B, 2,2, 3,9 e 5,4 </td></tr><tr><td> T4 DNA Ligase Buffer (NEB) </td><td> NEB </td><td> B0202S </td><td> Passo B, 3,3 e 3,9 </td></tr><tr><td> DNA polimerase de T4 </td><td> Promega </td><td> M4215 </td><td>Passo B, 1,2 </td></tr><tr><td> Quinase de polinucleótido T4 DNA </td><td> NEB </td><td> M0201 </td><td> Passo B, 3,3 </td></tr><tr><td> TruSeq SBS Kit v5-GA </td><td> Illumina </td><td> FC-104-5001 </td><td> Regentes para Illumina Genome Analyzer IIx sistema </td></tr><tr><td> TruSeq PE Cluster Kit v2-CBOT-GA </td><td> Illumina </td><td> PE-300-2001 </td><td> para ser usado com Illumina Sistema de Geração de Cluster CBOT </td></tr><tr><td> SYBR Green I </td><td> Invitrogen </td><td> S-7585 </td><td> Etapa B, 6,3 e 6,8 </td></tr><tr><td> XCell SureLock Sistema de Electroforese Mini-Cell </td><td> Invitrogen </td><td> EI0001 </td><td> Etapa B, 6,3 e 6,8 </td></tr></tbody></table><table border="1"><tbody><tr><td> Nome </td><td colspan="2"> Seqüência </td><td> Comentários </td></tr><tr><td rowspan="2"> Biotinilado meias-linkers A (200ng/μl) </td><td> Topo </td><td> 5 &#39;GG CCG CGA / iBiodT / ATC TTA TCC AAC 3&#39; </td><td> 250 escala nmole HPLC purificada Interna Biotina dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;TTG GGA TAA GAT ATC GC 3&#39; </td><td> 250 nmole HPLC escala purificada </td></tr><tr><td rowspan="2"> Biotinilado meias-linkers B (200ng/μl) </td><td> Topo </td><td> 5 &#39;GGC CGC GA / iBiodT / ATA CAT TCC AAC 3&#39; </td><td> 250 escala nmole HPLC purificada Interna Biotina dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;ATG GGA GTT TAT ATC GC 3&#39; </td><td> 250 nmole HPLC escala purificada </td></tr><tr><td rowspan="2"> Não biotinilado meias-linkers (200ng/μl) </td><td> Topo </td><td> 5 &#39;GGC CGC GAT ATC GGA TCC AAC 3&#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td> Bot </td><td> 5 &#39;TTG GGA TCC ATC GAT GC 3&#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td rowspan="2"> GS20 Adaptor A (200ng/μl) </td><td> Topo </td><td> 5 &#39;TCT CCA CAT CCC TGC GTG CAT TCCCTG TTC CCT CCC TGT CTC AGN N 3 &#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td> Bot </td><td> 5&#39;CTG AGA GGA GGG AAC CAG AGA TGG CAG GAC ACG GGA TGA GAT GG 3 &#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td rowspan="2"> GS20 Adaptador B (200ng/μl) </td><td> Topo </td><td> 5 &#39;GCA CTG AGA CAC ACA AGG GGG GAT CAA GGC CAG ACA GG GGG ATA 3&#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td> Bot </td><td> 5 &#39;ATC CCT CCC TGT GTG TGC CCT CTA TCC CCT GTT GCG TGT CTC AGN N 3&#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td rowspan="2"> Illumina-NN adaptadores </td><td> Topo </td><td> 5 &#39;CTC TTT ACA ACG CCC TAC ACG CTC TTC CGA TC 3&#39; </td><td> 250 nmole escala purificada por HPLC Veja Etapa B, 5,4 </td></tr><tr><td> Bot </td><td> 5 &#39;fosfato - GAT CGG GGT AAG AGC TCA ACG GGA ATG CCG AG 3&#39; </td><td> 250 nmole escala purificada por HPLC fosforilada 5 &#39;Veja Etapa B, 5,4 </td></tr><tr><td> Illumina 1-454 (frente) primer (10 uM) </td><td colspan="2"> 5 &#39;ACG AAT GAT GCG ACC ACC ACC GAG ATC TAC CTA TCC CCT GTG TGC CTT G 3&#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td> Illumina 2-454 primer (reverso) (10 mM) </td><td colspan="2"> 5 &#39;CAA ACG GAA GAC GGC ATA CGA CGG GAT ATC TCC ACT TCC CTG CGT GTC 3&#39; </td><td> 250 escala nmole PCR Grau </td></tr><tr><td> qPCR Primer 1,1 (10 uM) </td><td colspan="2"> 5 &#39;ACG AAT GAT GCG GAG ACC ACC AT 3&#39; </td><td> 10nmole escala PCR Grau </td></tr><tr><td> qPCR Primer 2,1 (10 uM) </td><td colspan="2"> 5 &#39;CAA ACG GAA GAC GGC ATA CGA 3&#39; </td><td> 10nmole escala PCR Grau </td></tr><tr><td> Illumina 3-454 iniciador de sequenciação (100 uM) </td><td colspan="2"> 5&#39;TGC GTG CAT TCC CTG TTC CCT CCC TGT CTC AG 3 &#39; </td><td> 100nmole CLAE em escala purificada </td></tr><tr><td> Illumina 4-454 iniciador de sequenciação (100 uM) </td><td colspan="2"> 5&#39;GTG CCT CTA TGC GTT GCG TCC CCT TGT CTC AG 3 &#39; </td><td> Escala 100nmoleHPLC purificada </td></tr></tbody></table> Oligos Tabela de oligos e adaptadores. Pedido de DNA integrado Technologies (IDT) e preparar linkers e adaptadores da mesma maneira como descrito anteriormente 10. Meia-linkers e adaptadores podem ser preparados de antemão e armazenados durante vários meses a -20 ° C.

chromatin interaction analysis with paired-end tag sequencing (chia-pet) for mapping chromatin interactions and understanding transcription regulation

Genomas são organizadas em estruturas tridimensionais, que adopta conformações de ordem mais elevada no interior dos espaços micron de tamanho nucleares 7, 2, 12. Tais arquiteturas não são aleatórias e envolvem interações entre genes promotores e elementos regulatórios 13. A ligação de fatores de transcrição específicos para seqüências regulatórias traz uma rede de regulação da transcrição e coordenação 1, 14. Cromatina Análise de Interação por Emparelhados Fim-Tag Sequencing (Chia-PET) foi desenvolvido para identificar estas estruturas de ordem superior de cromatina 5,6. As células são fixadas e loci que interagem entre são capturados por covalentes DNA-proteína ligações cruzadas. Para minimizar o ruído não-específica e reduzir a complexidade, bem como para aumentar a especificidade da interacção análise da cromatina, cromatina imunoprecipitação (ChIP) é usada contra factores proteicos específicos para enriquecer fragmentos de cromatina de interesse antes da ligadura proximidade. Ligadura envolvendo meias-linkers subsequentemente forma ligações covalentes entre os pares de fragmentos de DNA amarrados em conjunto dentro de complexos de cromatina individuais. Os sítios de restrição flanqueando CEMm enzimas nas meias-linkers permitir a extração de pares finais tag-tag-linker construções (PETS) sobre a digestão CEMm. À medida que os ligantes são meias-biotinilado, essas construções de PET são purificados utilizando estreptavidina-magnéticos grânulos. Os PETs purificados são ligados com adaptadores de seqüenciamento da próxima geração e um catálogo de fragmentos de interação é gerada através de próxima geração de sequenciadores como o Analyzer Genoma Illumina. Mapeamento e análise bioinformática é então realizada para identificar ChIP enriquecido locais de ligação e as interações ChIP enriquecidos com cromatina 8. Produzimos um vídeo para demonstrar aspectos críticos do protocolo Chia-PET, especialmente a preparação de ChIP como a qualidade do ChIP desempenha um papel importante no resultado de uma biblioteca de Chia-PET. À medida que os protocolos sãomuito longo, apenas os passos críticos são mostrados no vídeo.

Watch this Scientific Journal Video about Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation at JoVE.c

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

Cromatina Análise de Interação por Emparelhados Fim-Tag Sequencing (Chia-PET) é um método para<em&gt; De novo</em&gt; Detecção de interações de cromatina, para melhor entendimento do controle da transcrição.

Cromatina Análise de Interação com Emparelhados Fim-Tag Sequencing (Chia-PET) para mapeamento Interações cromatina e Compreensão regulação da transcrição

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

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Research

JoVE Journal

Biology

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

30.5K visualizações.  Agency for Science, Technology and Research, Singapore. Cromatina Análise de Interação por Emparelhados Fim-Tag Sequencing (Chia-PET) é um método para De novo Detecção de interações de cromatina, para melhor entendimento do controle da transcrição.

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

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Um Ensaio Chromatin para tecido do cérebro humano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Biologia

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Leia única e emparelhados End mRNA-Seq Bibliotecas Illumina de 10 nanogramas RNA total

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Cromatina imunoprecipitação (CHIP), utilizando Drosophila Tecido

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Isolamento de cromatina por purificação de RNA (CHIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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

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

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

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

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

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

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Um método eficiente para Quantitativa, Análise de células Único de cromatina Modificação e Arquitetura Nuclear em Todo-mount óvulos em<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

Geração e Purificação de INO80 cromatina remodelação Complexos Humanos e subcomplexos

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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

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

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

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

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<em&gt; In Vivo</em

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

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).