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E. Zambrano, P. M. Martínez-samayoa, G. L. Rodríguez-gonzález, and P. W. Nathanielsz, « RAPID REPORT: Dietary Intervention Prior to Pregnancy Reverses Metabolic Programming in Male Offspring of Obese Rats: Dietary Intervention to Reverse Metabolic Programming Outcomes, The Journal of Physiology, vol.588, issue.10, pp.1791-99, 2010.

E. E. Zhang, Y. Liu, R. Dentin, Y. Pagkapol, . Pongsawakul et al., « Cryptochrome Mediates Circadian Regulation of CAMP Signaling and Hepatic Gluconeogenesis », Nature Medicine, vol.16, issue.10, pp.1152-56, 2010.

. Zhang, J. L. Song, A. Morrison, L. Gill, . Rattanatray et al., « Maternal Dietary Restriction During the Periconceptional Period in Normal-Weight or Obese Ewes Results in Adrenocortical Hypertrophy, an Up-Regulation of the JAK/STAT and Down-Regulation of the IGF1R Signaling Pathways in the Adrenal of the Postnatal Lamb, Endocrinology, vol.154, issue.12, pp.4650-62, 2013.

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D. , , vol.29, pp.2646-2681

N. , , vol.63, pp.56353-56368, 1121.

, Ajouter de l'inhibiteur de protéase (fourni dans le kit) extemporanément. A refroidir sur glace avant usage, Tampon TE : Tris-EDTA 1X PBS : Ajouter de l'inhibiteur de protéases avant usage. Pierce TM Methanol-free Formaldehyde Ampules

. Formaldehyde, Methanol-Free 28906

, Préparation des tissus (temps dépend du tissus) et Cross-Linking (~2h)

, Sous hotte : préparer le formaldéhyde 1% à partir du formaldéhyde 16% ? Formaldéhyde (16%) 62,5 µl + 937,5 µl PBS

, Broyer les tissus dans de l'azote liquide. Et transférer la poudre dans des tubes de 2 mL préalablement annotés

, Ajouter rapidement 1 ml de Formaldéhyde 1% (1 ml pour 30mg de tissus en général. Jusqu'à 300mg pour du tissus adipeux et 15 mg pour du tissus musculaire)

, Inverser le tube plusieurs fois immédiatement après l'ajout

, Incuber le tube à température ambiante sur rotateur/roue pendant 8 minutes (ce temps peut varier en fonction du type cellulaire, une optimisation peut être nécessaire)

, Stopper le crosslinking en ajoutant 100 µl de glycine (contenue dans le kit) et incuber le tube sur roue pendant 10 minutes

, Centrifuger a 2000g pendant 10 minutes à 4°C pour culotter les tissus

, Remuer le tube en le heurtant d'un doigt plusieurs fois pour décrocher le culot. Si le culot a du mal à se décrocher

, Culoter les tissus en centrifugeant à 2000g pendant 10 minutes à 4°C

, Remuer le tube en le heurtant d'un doigt plusieurs fois pour décrocher le culot. Si le culot a du mal à se décrocher

, Culotter les tissus en centrifugeant à 2000g pendant 10 minutes à 4°C et éliminer le surnageant. Le culot de tissus crosslinké peut ensuite être refroidi dans de l'azote liquide pour ensuite être stocké à -80°C si nécessaire

, Extraction de Chromatine et Cisaillement 10

V. Protocole, ChIP-seq, basé sur le protocole utilisé au département des Sciences Animales de l

, Incuber le tube sur glace pendant 20 minutes. (Cette incubation sert à faire gonfler les cellules pour permettre la lyse mécanique, tout en laissant les noyaux intacts

, Transférer le tissu en suspension dans un dounce préalablement refroidi et homogénéiser en faisant 30-40 aller-retour de pilon

, Transférer l'homogénat dans un tube neuf de 2 ml (ou 5 ml en fonction du tissus) puis rincer le dounce avec 500 µl de iL1 Buffer et combiner avec le tube de 2 ml

, Culoter les noyaux en centrifugeant à 2000g pendant 5 minutes à 4°C. (Ce culot ne sera pas collé au tube autant que le premier et sera principalement blanc

, Eliminer le surnageant et resuspendre le culot dans 2 ml de iL2 Buffer

, Culoter les noyaux en centrifugeant à 2000g pendant 5 minutes à 4°C

, Eliminer le surnageant et resuspendre dans 660µl de iS1 Buffer

, Prélever un aliquote de 5µl de chaque échantillon et le diluer dans 15 µl de TE 1X pour un gel analytique. Conserver les aliquotes dans la glace jusqu'à utilisation. Ces aliquotes sont là pour confirmer que la chromatine était intacte avant sonication

, Protocole de sonication dépendant de l'appareil utilisé

, Une fois la sonication complète, enlever les échantillons du sonicateur et les conserver dans la glace

, Prélever un aliquot de 5 µl de chaque tube soniqué et les diluer dans 45µl de TE 1X pour un gel analytique

, Traiter les aliquots par 20 µg (2µl) de Proteinase K pendant 30 minutes à 65°C puis traiter à la RNAse A (2µl (10mg/ml))

, Ou ajouter 6 µl de NaCl 5M aux 50 µl et incuber 30 minutes à 95°C. Ces étapes servent à dégrader proteines et ARNs dans le premier cas ou forcer le décrosslinking dans le second cas. Si la seconde méthode est choisie

, Prélever 10 µl et ajouter un colorant de charge à chacun des échantillons puis les faire migrer sur un gel d'agarose 1

, Visualiser les échantillons d'ADN sur le gel d'agarose. (La Chromatine cisaillée doit se trouver dans une fourchette de taille de 200 à 500 pb

, A ce point, il est possible de congeler et stocker à -80°C les échantillons soniqués pour une utilisation ultérieure (cf

, Si l'utilisation est dans peu de temps, il est possible de les conserver à -20°C

, Pour chacun des aliquotes de chromatine soniquée précédemment constitués, ajouter 54 µl de TE 1X (Vf=100µl)

, Mesurer la concentration grâce au Nanodrop et estimer la concentration initiale de la chromatine soniquée. Cette concentration sera utilisée pour les calculs nécessaires à l'ImmunoPrécipitation

, Penser à sortir les réactifs/solutions et faire décongeler sur glace. L'isopropanol doit être à RT. Les billes (IPure Beads v2) doivent être conservées sur glace (pour éviter qu'elles sèchent) et vortexées avant chaque utilisation

, Pooler les échantillons si nécessaire. 2 échantillons peuvent facilement être poolés. Si plus de 2 échantillons doivent l'être, procéder à la purification de chaque échantillon individuellement, pooler les éluats à la fin de la purification et concentration iPure

, Ajouter 100 µl d'isopropanol 100% à chaque IP et INPUT et inverser les tubes plusieurs fois pour mélanger. La solution peut devenir trouble, cela n'a pas d'incidence sur la qualité ou la quantité d'ADN purifié

, Placer les tubes sur roue/rotateur au fur et à mesure. Déclencher le chronomètre après le dernier tube et incuber à RT pendant 10 minutes sur roue à 40 rpm, Resuspendre les billes fournies (IPure Beads v2) et transférer 10 µl dans chaque IP et INPUT

, Préparer les Wash buffer 1 et 2 contenant 50% d'isopropanol (1 volume de wash buffer pour un volume d'isopropanol 100%) Ne jamais laisser les bouteilles ouvertes pour éviter l'évaporation

, Placer ensuite les tubes sur le portoir magnétique. Attendre 1 minute, les billes doivent s'être rassemblées

, Ajouter 100 µl de Wash buffer 1 par tube puis vortexer pour remettre les billes en suspension (ou les placer sur rotateur en attendant l'incubation) puis centrifuger rapidement pour rassembler le liquide et incuber 1 minute sur la paillasse a RT, Fermer les tubes (pour éviter l'évaporation) et les retirer du portoir magnétique

, Centrifuger rapidement les tubes pour rassembler le liquide si nécessaire puis les placer sur portoir magnétique, attendre au moins 1 minute et éliminer le surnageant

, Ajouter 100 µl de Wash buffer 2 par tube. Vortexer ensuite les tubes pour remettre les billes en suspension (ou les placer sur rotateur en attendant l'incubation) puis centrifuger rapidement pour rassembler le liquide et incuber 1 minute sur la paillasse a RT, Fermer les tubes et les retirer du portoir magnétique

, Centrifuger rapidement les tubes pour rassembler le liquide si nécessaire puis les placer sur portoir magnétique, attendre 1 minute et éliminer le surnageant. Centrifuger de nouveau les tubes et les remettre sur portoir magnétique puis enlever le reste de Wash buffer 2 si nécessaire

, Resuspendre les billes avec 55 µl de Buffer C, mélanger à la pipette (5-6 va et viens) et incuber à température ambiante sur roue à 40 rpm pendant 15 minutes

, Pendant l'incubation : Préparer tubes et réactifs pour quantification QBit

, Puis à l'aide d'une P2 prélever 2 µl. Centrifuger de nouveau les tubes contenant les billes et les replacer sur portoir magnétique, essayer de récupérer le reste de liquide, sans aspirer de billes, Centrifuger les tubes pour rassembler le liquide et les placer sur portoir magnétique

, Prélever 2 µl d'ADN ImmunoPrécipité et les mettre dans les tubes préalablement préparés pour la quantification au QBit. Ne pas les diluer, la concentration est généralement faible à cette étape

, Prélever 42 µl d'éluât et le transférer dans un nouveau tube de PCR préalablement annote

, Pour cela prélever 2 µl de l'éluât, le disposer dans un nouveau tube de PCR contenant au moins 4 µl de TE 0.1X. Passer les librairies diluées au Qbit pour s'assurer de leur concentration, de préférence la concentration doit être inférieure à 20 ng/µl pour être utilisée dans le Bioanalyzer, Vérifier la distribution des fragments d'ADN à l'aide d'une Puce ADN High Sensitivity sur Bioanalyzer d'Agilent

, Sur le Bioanalyzer le plus gros des pics doit être a la taille attendue des fragments ex : ~300 pour librairie de

, Si des fragments plus gros sont présents ce n'est pas grave tant que la molarité du grand pic leur est très supérieure

, Si le grand pic présente un gap c'est que l'échantillon analyse est trop concentre, ajouter quelques µl de TE 0

, Les librairies doivent être stockées à -80°C