Les auteurs sont pris en charge par A * STAR de Singapour. En outre, MJF est soutenu par une bourse d&#39;études A * STAR National Science, un Oréal For Women in Science National Fellowship et un Lee Kuan Yew de postdoctorat. YR est soutenu par le NIH ENCODE subventions (R01-01 et HG004456 R01 HG003521-01). Les auteurs remercient également l&#39;équipe de la vidéographie de Productions Pixels 8, Singapour, en particulier Issey M. Kelvin, M. Chang Kai Xiang et M. Sherwin Gan pour le tournage des scènes, Mme Siti Rahim pour le montage vidéo et de Mme Michelle Teo pour le voice-over.

<ol>
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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. 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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>

Pas de conflits d&#39;intérêt déclarés.

Chia-PET est une méthode mise au point pour identifier les interactions à longue portée dans régulation de la transcription. Un des facteurs critiques qui déterminent la qualité d&#39;une bibliothèque Chia-PET est la qualité du matériel ChIP. Le protocole présenté dans la vidéo incorpore l&#39;utilisation de biens et services environnementaux et de formaldéhyde pour réticuler les cellules. L&#39;utilisation du formaldéhyde combiné avec un deuxième réactif de réticulation portant un bras espaceur plus peut aider à la fixation de protéines qui ne pouvaient pas être liés par le formaldéhyde seul 3,11,15. Nous avons construit des bibliothèques avec cette méthode, qui ont démontré de solides sites de liaison et à long terme des interactions 9. Toutefois, la réticulation et les conditions de puce devrait être optimisé pour chaque facteur d&#39;intérêt, et il est important de ne pas trop réticulation comme trop réticulation se traduira par des difficultés dans la fragmentation par ultrasons, et peut éventuellement mener à des interactions parasites chromatine . Interactions chromatineidentifié par Chia-PET doit être validé par une méthode différente, telles que la fluorescence par hybridation in situ 4. Nous recommandons un minimum de 100 ng de matériel chromatine. Alors que nous avons construit des bibliothèques de bonne qualité à partir de 50 ng de matériel chromatine, nous avons observé que de grandes quantités de matériau de départ a permis la construction de Chia-PET bibliothèques avec des cycles de PCR de moins de 16, réduisant ainsi au minimum amplicons et la redondance de chaque bibliothèque. Cette baisse de la redondance en corrélation avec plus uniques balises cartographiés et également un pourcentage élevé de données utilisables, ce qui permet une carte plus complète chromatine interaction avec moins de voies de séquençage. Le volume final emballé de perles dans chaque tube devrait être de 50 pi et 100 pi de billes magnétiques et Sépharose respectivement. Si le volume emballé perle est inférieure à celle indiquée, mettre le volume au minimum emballé avec même pré-autorisé vierges billes magnétiques ou Sepharose pour minimiser la perte de l&#39;ADN porteur de billess dans les étapes ultérieures. À canon scié de conseil ou grand-core des conseils devraient être utilisés pour le pipetage des billes de Sépharose. Les modifications suivantes ont été intégrées à la suite du déjà publié Chia-PET protocole 5. Tout d&#39;abord, billes magnétiques G ont été utilisés pour minimiser la perte de l&#39;échantillon pendant les lavages. En outre, nous avons identifié bandes non spécifiques avec la taille approximative de 100 pb et 138 pb pour être amplicons de l&#39;auto-ligaturés demi-linkers ou / et des adaptateurs. Par conséquent, nous avons réduit la concentration de biotinylés demi-linkers et 454 GS20 adaptateurs pour minimiser bandes non spécifiques lors de l&#39;amplification PCR. Le volume ligature de proximité a été réduit de 50 ml à 10 ml pour minimiser la perte d&#39;échantillon au cours des étapes ultérieures de purification et aussi économiser sur les coûts des réactifs. Nous avons également augmenté le temps d&#39;incubation pour immobiliser Chia-PET ADN à des billes pour assurer la capture maximale de Chia-PET ADN sur les billes de streptavidine. Au cours de l&#39;étape de ligation de proximité, la ligature des chimères that ne représentent pas vrai dans les interactions in vivo chromatine sont inévitablement générés d&#39;une manière non-spécifique et aléatoire. Par conséquent, pour évaluer la qualité des données provenant de n&#39;importe quelle expérience Chia-PET, le taux de chimérisme est estimée à partir de l&#39;utilisation de deux différents demi-linkers avec nucléotidique spécifique TAAG codes à barres et ATGT 5. Après séquençage à haut débit, les séquences Chia-PET sont d&#39;abord analysés pour leur composition code-barres de liaison et des séquences dérivées de produits de ligature et des produits spécifiques ligature non-spécifiques peuvent être distinguées 8. Le pourcentage de chimères connus (c.-à-hétérodimères linkers AB) présents dans notre propre MCF-7 ARN polymérase II Chia-PET bibliothèques est inférieure à 15%. Chia-PET séquences sont ensuite classées en deux catégories, à savoir l&#39;auto-ligature animaux de compagnie et inter-ligature ANIMAUX DE COMPAGNIE. Auto-ligature PET sont obtenues à partir d&#39;auto-circularisation ligature des fragments de chromatine tandis entre ligature PET sont dérivées à partir de inter-ligature entre deux fragments d&#39;ADN différents. Ce dernier est alors sous-divisé en trois catégories différentes en fonction de la distance génomique de chaque étiquette sur le même chromosome (intrachromosomiques inter-ligature PET) ou que les deux balises sont mappées à deux chromosomes différents (interchromosomiques inter-ligature PET). Nous avons développé un package Chia-PET outil logiciel pour trier les différentes catégories 8. Ce sera basé sur des fragments d&#39;ADN qui sont dans la bibliothèque. En règle générale, des fragments plus petits ChIP donnera une résolution plus élevée et la couper pour ces ARN polymérase II Chia-PET bibliothèques est d&#39;environ 4 ko. En outre, les interactions réelles chromatine peuvent être distingués du bruit aléatoire en comptant le nombre d&#39;inter-ligature vie privée dans un cluster interaction; en d&#39;autres termes, un groupe de comptage PET haute est dit d&#39;avoir une plus grande probabilité d&#39;être une interaction réelle chromatine 8 . Pour filtrer nos faux positifs unepassera de points d&#39;ancrage hautement enrichi qui peuvent former des inter-ligature vie privée par hasard, un cadre d&#39;analyse statistique a également été formulées pour tenir compte de la formation aléatoire de toute vie privée inter-ligature entre deux ancres 8. En conclusion, la technique de Chia-PET permet de cartographier les réseaux d&#39;interaction chromatine à l&#39;échelle mondiale. La mise en œuvre de la puce en Chia-PET permet la réduction de la complexité de bibliothèque et le bruit de fond. En outre, ajoute-t-ChIP spécificité des interactions chromatine, permettant l&#39;examen des interactions spécifiques associés à la chromatine facteurs de transcription particuliers 5.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Gradient 4-20% TBE gel </td><td> Invitrogen </td><td> EC6225BOX </td><td> Étape B, 6,3 </td></tr><tr><td> 5 x tampon T4 ADN ligase avec le PEG </td><td> Invitrogen </td><td> 46300018 </td><td> Etape B, 2,2 </td></tr><tr><td> 6% de gel TBE </td><td> Invitrogen </td><td> EC6263BOX </td><td> Étape B, 6,8 </td></tr><tr><td> Agilent ADN 100 Assay </td><td> Agilent Technologies </td><td> 5067-1504 </td><td rowspan="3"> Etape B, 7,1 </td></tr><tr><td> Agilent test ADN de haut Sensibilité </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> Centrifuger Filtres Tube (Spin-X) </td><td> Corning &lt;/ Td&gt; <td> CLS8160 </td><td> Étape B, 3,7 et 6,9 </td></tr><tr><td> Transilluminateur lecteur foncé </td><td> Clare Chemical Research </td><td> DR46B </td><td> Étape B, 6,8 </td></tr><tr><td> Numérique portable Sonifier Disrupter </td><td> Branson </td><td> 450D-0101063591 </td><td> Étape A, 4,3 </td></tr><tr><td> Dynamag-2 aimant (concentrateur de particules magnétiques) </td><td> Invitrogen </td><td> 123-21D </td><td> Étape A, B, étape 7.5.1, pour tous les billes magnétiques des étapes de lavage </td></tr><tr><td> Dynamag-15 Aimant (concentrateur de particules magnétiques) </td><td> Invitrogen </td><td> 123.01D </td><td> Etape A, 5 et 6 </td></tr><tr><td> Dynamag-PCR (concentrateur de particules magnétiques) </td><td> Invitrogen </td><td> 49-2025 </td><td> Étape B, 6,5 </td></tr><tr><td> Polymérase ADN d&#39;Escherichia coli </td><td> ONÉ </td><td> M0209 </td><td> Étape B, 5,6 </td></tr><tr><td> GlycoBlue </td><td> Ambion </td><td> AM9516 </td&gt; <td> Étape A, B Étape 7.5.1, 4.3 et 6.5 </td></tr><tr><td> Illumina cbot Generation System Cluster </td><td> Illumina </td><td> SY-301-2002 </td><td> Etape B, 7,4 </td></tr><tr><td> Illumina Genome Analyzer IIx </td><td> Illumina </td><td> SY-301-1301 </td><td> Etape B, 7,5 </td></tr><tr><td> Illumina PE amorces </td><td> Illumina </td><td> PE-102-1004 </td><td> Etape B, 5.4 (si nécessaire) </td></tr><tr><td> Intelli-Mixer </td><td> Palico Biotech </td><td> RM-2L </td><td> Étape B, toutes les incubations avec rotation </td></tr><tr><td> LightCycler 480 Real-Time System PCR </td><td> Roche </td><td> 04 640 268 001 </td><td> Étape A, B 7.5.3 Étape, 7,3 </td></tr><tr><td> LightCycler480 ADN SYBR Green I MasterMix </td><td> Roche </td><td> 03 752 186 001 </td><td> Etape B, 7.5.3 </td></tr><tr><td> M-280 Streptavidin Dynabeads </td><td> Invitrogen </td><td> 11206D </td><td> Étape B, 5,2 </td></tr> &lt;tr&gt; <td> Magnétique protéines (Dynabeads) G </td><td> Invitrogen </td><td> 100.03D </td><td> Étape A, 5.1.1 et 5.2.1 </td></tr><tr><td> MaXtract haute densité (2ml) </td><td> Qiagen </td><td> 129056 </td><td> Étape A 7.5.1, </td></tr><tr><td> MaXtract haute densité (50ml) </td><td> Qiagen </td><td> 129073 </td><td> Etape B, 4,2 </td></tr><tr><td> MmeI </td><td> ONÉ </td><td> R0637 </td><td> R0637 </td></tr><tr><td> RNase ONE Ribonuclease </td><td> Promega </td><td> M426C </td><td> Etape B, 4,8 </td></tr><tr><td> L&#39;ARN polymérase II (8WG16) anticorps monoclonal </td><td> Covance </td><td> MMS-126R </td><td> Étape A, 5.2.4 </td></tr><tr><td> Tubes à centrifuger Oak Ridge (polypropylène) </td><td> Nalgene </td><td> 3119-0050 </td><td> Étape A, 3,1 </td></tr><tr><td> Tubes à centrifuger Oak Ridge (Téflon FEP) </td><td> Nalgene </td><td> 3114-0050 </td><td> Etape B, 4,3 </td></tr><tr><td> Adolpheion haute fidélité Master Mix </td><td> Finnzymes </td><td> F-531 </td><td> Étape B, 6 </td></tr><tr><td> Picogreen (Quant-iT) Réactif ADNdb </td><td> Invitrogen </td><td> P11495 </td><td> Étape A, 7.5.2 </td></tr><tr><td> Polystyrène Tube ronde test Bas </td><td> BD Biosciences </td><td> 352057 </td><td> Etape A, 4,1 </td></tr><tr><td> Comprimés inhibiteurs de la protéase à cocktails (complet, EDTA-free) </td><td> Roche </td><td> 11873580001 </td><td> Étape A partir de 1.6, </td></tr><tr><td> Protéinase K Solution (20mg/ml) </td><td> Fermentas </td><td> E00491 </td><td> Étape A, 4.4, 7.5.1 Etape B, 4,1 </td></tr><tr><td> ADN ligase T4 </td><td> Fermentas </td><td> EL0013 </td><td> Etape B, 2,2, 3,9 et 5,4 </td></tr><tr><td> T4 ADN ligase tampon (ONE) </td><td> ONÉ </td><td> B0202S </td><td> Etape B, 3,3 et 3,9 </td></tr><tr><td> ADN polymérase T4 </td><td> Promega </td><td> M4215 </td><td>Etape B, 1,2 </td></tr><tr><td> T4 polynucléotide kinase ADN </td><td> ONÉ </td><td> M0201 </td><td> Étape B, 3,3 </td></tr><tr><td> TruSeq SBS Kit v5-GA </td><td> Illumina </td><td> FC-104-5001 </td><td> Regents pour Illumina Genome Analyzer IIx système </td></tr><tr><td> TruSeq PE Cluster Kit v2-CBOT-GA </td><td> Illumina </td><td> PE-300-2001 </td><td> à utiliser avec Illumina cbot Generation System Cluster </td></tr><tr><td> SYBR Green I </td><td> Invitrogen </td><td> S-7585 </td><td> Etape B, 6,3 et 6,8 </td></tr><tr><td> XCell SureLock Système d&#39;électrophorèse Mini-Cell </td><td> Invitrogen </td><td> EI0001 </td><td> Etape B, 6,3 et 6,8 </td></tr></tbody></table><table border="1"><tbody><tr><td> Nom </td><td colspan="2"> Séquence </td><td> Commentaires </td></tr><tr><td rowspan="2"> Biotinylé demi-lieurs A (200ng/μl) </td><td> Haut </td><td> 5 &#39;GG GCC CGA / iBiodT / ATC TTA STC AAC 3&#39; </td><td> 250 nmoles échelle purifiée par HPLC interne Biotine dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA TAA GAT ATC GC 3&#39; </td><td> 250 nmoles échelle purifiée par HPLC </td></tr><tr><td rowspan="2"> Biotinylé demi-linkers B (200ng/μl) </td><td> Haut </td><td> 5 &#39;GGC CCG GA / iBiodT / ATA CAT TCC CAA 3&#39; </td><td> 250 nmoles échelle purifiée par HPLC interne Biotine dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA ATG TAT ATC GC 3&#39; </td><td> 250 nmoles échelle purifiée par HPLC </td></tr><tr><td rowspan="2"> Non-biotinylés demi-linkers (200ng/μl) </td><td> Haut </td><td> 5 &#39;GGC CCG CAG ATC GGA TCC CAA 3&#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA TCC CAG ATC GC 3&#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td rowspan="2"> GS20 Adaptateur A (200ng/μl) </td><td> Haut </td><td> 5 &#39;CCA TCT CAT CCC GTG TGC TCC CATCTG TTC CCT CCC TGT CCT AGN N 3 &#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td> Bot </td><td> 5&#39;CTG AGA GGA GGG CAG AAC AGA TGG GAC ACG ACG GGA GAT TGA GG 3 &#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td rowspan="2"> GS20 Adaptateur B (200ng/μl) </td><td> Haut </td><td> 5 &#39;CTG AGA CAC GCA ACA GGG GAT AGG CAA GGC CAG ACA GGG GG ATA 3&#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td> Bot </td><td> 5 &#39;ATC CCC CCT TGT GTG CCT TGC CTA TCC CCT GCG GTT TGT CCT AGN N 3&#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td rowspan="2"> Illumina-NN adaptateurs </td><td> Haut </td><td> 5 &#39;ACA CCT TTT CCC ACG ACG TAC CCT TTC CGA TC 3&#39; </td><td> 250 nmoles échelle purifiée par HPLC Voir l&#39;étape B, 5,4 </td></tr><tr><td> Bot </td><td> 5 &#39;phos - GAT CGG AAG CAG GGT TCA GCA GGA ATG GCC AG 3&#39; </td><td> 250 nmoles échelle purifiée par HPLC phosphorylée extrémité 5 &#39;Voir l&#39;étape B, 5,4 </td></tr><tr><td> Illumina 1-454 (en avant) amorce (10 uM) </td><td colspan="2"> 5 &#39;AAT GAT GCG ACG ACC ACC ACC GAG ATC TAC TCC CTA GTG CCT TGC CTT G 3&#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td> Illumina 2-454 (inverse) amorce (10 uM) </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC GAT CGG ATA CGA TCC ATC TCA TCC CTG CGT GTC 3&#39; </td><td> 250 nmoles échelle PCR grade </td></tr><tr><td> Primer qPCR 1.1 (10 uM) </td><td colspan="2"> 5 &#39;AAT GAT ACG GCG CAC GAG ACC À 3&#39; </td><td> 10nmole échelle PCR grade </td></tr><tr><td> Primer qPCR 2.1 (10 uM) </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC CGA ATA 3&#39; </td><td> 10nmole échelle PCR grade </td></tr><tr><td> Illumina 3-454 amorce de séquençage (100 uM) </td><td colspan="2"> 5&#39;TGC GTG TCC CAT CTG TTC CCT CCC TGT CCT AG 3 &#39; </td><td> 100nmole échelle purifiée par HPLC </td></tr><tr><td> Illumina 4-454 amorce de séquençage (100 uM) </td><td colspan="2"> 5&#39;GTG CCT TGC CTA TCC CCT GCG GTT TGT CCT AG 3 &#39; </td><td> Échelle 100nmolePurifiée par HPLC </td></tr></tbody></table> Oligos commander les Tables des oligos et adaptateurs. De Integrated DNA Technologies (IDT) et de préparer les linkers et adaptateurs de la même manière que décrit précédemment 10. Demi-linkers et adaptateurs peut être préparé à l&#39;avance et stockés pendant plusieurs mois à -20 ° C.

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

Les génomes sont organisés en structures tridimensionnelles, l&#39;adoption d&#39;un ordre supérieur conformations à l&#39;intérieur des espaces de taille micronique nucléaires 7, 2, 12. De telles architectures ne sont pas aléatoires et font intervenir des interactions entre les promoteurs des gènes et des éléments régulateurs 13. La liaison des facteurs de transcription à des séquences régulatrices spécifiques provoque un réseau de régulation de la transcription et la coordination 1, 14. Analyse de l&#39;interaction de la chromatine par Tag appariés Fin de séquençage (Chia-PET) a été développé pour identifier ces structures chromatiniennes d&#39;ordre supérieur 5,6. Les cellules sont fixées et en interaction locus sont capturés par covalentes ADN-protéine liaisons transversales. Pour minimiser la non-spécifique du bruit et de réduire la complexité, ainsi que d&#39;augmenter la spécificité de l&#39;analyse de l&#39;interaction de la chromatine, immunoprécipitation de la chromatine (ChIP) est utilisé contre les facteurs protéiques spécifiques pour enrichir des fragments de chromatine d&#39;intérêt avant la ligature de proximité. La ligature impliquant demi-linkers fait suite liaisons covalentes entre des paires de fragments d&#39;ADN attachés ensemble au sein de complexes chromatiniens individuels. Les sites d&#39;accompagnement MmeI enzymes de restriction dans les linkers demi-permettre l&#39;extraction de paires de balise de fin-linker-tag constructions (PET) sur la digestion MmeI. Comme les linkers sont des demi-biotinylé, ces constructions en PET sont purifiés en utilisant la streptavidine-billes magnétiques. Les PET purifiés sont ligués avec des adaptateurs de séquençage de nouvelle génération et un catalogue des fragments d&#39;interaction est généré par l&#39;intermédiaire de la prochaine génération des séquenceurs tels que l&#39;analyseur de Génome Illumina. Cartographie et analyse bioinformatique est ensuite réalisée afin d&#39;identifier ChIP-enrichi sites de liaison et de ChIP-enrichis interactions chromatine 8. Nous avons produit une vidéo pour démontrer les aspects critiques du protocole de Chia-PET, en particulier la préparation de la puce comme la qualité de ChIP joue un rôle majeur dans l&#39;issue d&#39;une bibliothèque Chia-PET. Comme les protocoles sonttrès longtemps, seules les étapes critiques sont montrées dans la vidéo.

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

Analyse de l&#39;interaction de la chromatine par Tag appariés Fin de séquençage (Chia-PET) est une méthode pour<em&gt; De novo</em&gt; Détection des interactions chromatine, pour une meilleure compréhension du contrôle de la transcription.

Analyse de l&#39;interaction avec la chromatine Tag appariés Fin de séquençage (Chia-PET) pour la cartographie des interactions Chromatine et transcription Comprendre le règlement

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

chromatin-interaction-analysis-with-paired-end-tag-sequencing-chia

Research

JoVE Journal

Biology

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

30.5K vues.  Agency for Science, Technology and Research, Singapore. Analyse de l'interaction de la chromatine par Tag appariés Fin de séquençage (Chia-PET) est une méthode pour De novo Détection des interactions chromatine, pour une meilleure compréhension du contrôle de la transcription.

Vidéo: 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.

Un dosage de la chromatine pour les tissus du cerveau humain

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

Recherche

Biologie

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.

Simple Lire et jumelés bibliothèques Fin Illumina ARNm-Seq de 10 nanogrammes d&#39;ARN 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.

Immunoprécipitation de la chromatine (ChIP) à l&#39;aide Drosophila Tissus

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.

Isolement de la chromatine par purification d&#39;ARN (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.

Génération de haute qualité Immunoprécipitation de la chromatine Patron de l&#39;ADN pour le séquençage à haut débit (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.

Une méthode efficace pour quantitative, analyse unicellulaire de la chromatine Modification et architecture nucléaire dans l&#39;ensemble du montage ovules dans<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.

Production et purification des droits de INO80 remodelage de la chromatine Complexes et Subcomplexes

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.

Immunoprécipitation de la chromatine test pour l&#39;identification de Arabidopsis interactions protéine-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.

Isolement des régions génomiques spécifiques et l&#39;identification des molécules associées par 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).

La cartographie du génome à l&#39;échelle du médicament-ADN interactions dans les cellules avec COSMIC (réticulation de petites molécules pour isoler la chromatine)