Bu çalışma Takeda Bilim Vakfı (TF) tarafından desteklenmiştir; Asahi Glass Vakfı; Uehara Memorial Foundation (HF); Kurata Memorial Hitachi Bilim ve Teknoloji Vakfı (TF ve HF); Grant-Aid Genç Bilginler (B) (# 25830131), Grant-Aid Bilimsel Araştırma (C) (# 15K06895) (TF) için; ve Hibe-Aid Yenilikçi Alanları &#39;Transkripsiyon Döngüsü&#39; Bilimsel Araştırma (# 25118512 &amp; # 15H01354), Grant-Aid Bilimsel Araştırma (B) (# 15H04329) için, Grant-Aid Keşif Araştırma ( # 26650059) ve Japonya Eğitim Bakanlığı, Kültür, Spor, Bilim ve Teknoloji dan &#39;Genom Desteği&#39; (# 221S0002) (HF).

Hedef lokusun tanıma Engineered DNA-bağlama Moleküllerin 1. Tasarım <ol><li> CRISPR kompleksleri kullanılarak enChIP için, daha önce, 18 olarak tanımlanan ilgili genomik bölgenin aday gRNA hedef sekanslarını tespit etmek CRISPRdirect web aracı (http://crispr.dbcls.jp) kullanın. Bu web aracı formunun 23 bp genomik siteleri döndürür 5&#39;-N 20 NGG-3 &#39;hedef bölge içinde. </li><li> U6 promotör sekansı ve 5&#39;-N 20 sekansına ticari hizmetlerin kullanılarak 5&#39;-N 20 NGG-3 &#39;içeren bir gBlock sentezlenmesi 19,20 (bakınız Şekil 2). Amaçlara alt klonlama için gBlock dışındaki uygun kısıtlayıcı enzim sitelerinin içerir. </li><li> Daha önce tarif edildiği gibi 16, uygun bir vektör içine gBlock yerleştirin. GBlocks için çeşitli retroviral vektörler (Malzeme) mevcuttur. </li><li> TAL proteinler kullanılarak enChIP için Targ tanıyan TAL proteinleri tasarımet loci. Ticari hizmetlerini kullanarak TAL proteinleri kodlayan plazmid oluşturun. Daha önce tarif edildiği gibi 15 3 x FLAG bir veya daha fazla etiketleri ile kaynaşık TAL proteinleri ihtiva eden ifade vektörlerini oluşturmak. </li></ol> EnChIP Analizi Hücre 2. kurulması <ol><li> 3 x FLAG-dCas9 ve gRNA (CRISPR bazlı prosedür) ya da Express 3, daha önce tarif edildiği gibi 14-17 x FLAG-TAL hücrelerinde (TAL protein bazlı prosedür) analiz edilecek. <ol><li> Transfeksiyon verimi yüksek ise geçici transfeksiyon kullanın. 3 x FLAG-dCas9 CMV promotörü ihtiva eden bir ekspresyon vektörü (Malzeme) mevcuttur. </li><li> Geçici transfeksiyon verimliliği düşükse geleneksel yöntemler kullanılarak stabil transformantların kurulması düşünün. GBlock için yukarıda belirtilen retroviral vektörler ek olarak, 3 x FLAG-dCas9 retroviral vektörler (Malzeme) mevcuttur. </li></ol></li><li&gt;, Daha önce tarif edildiği 14-17 gibi bir anti-FLAG antikoru (Ab) kullanılarak bağışıklık beneklenme analizi ile transfekte edilmiş hücrelerde 3xFLAG-dCas9 ya 3xFLAG-TAL proteininin ifadesini teyit edin. Not: Bu etiketli proteinlerinin ekspresyonu, aynı zamanda, anti-FLAG-FITC ve daha sonraki bir FACS analizi ile hücre içi boyama ile teyit edilebilir. Hücre içi boyama için bir protokol bizim ana (http://www.biken.osaka-u.ac.jp/lab/microimm/fujii/iChIP_protocols/english.html) indirilebilir. </li><li> Standart RT-PCR tekniği ile gRNA ifadesini onaylayın. </li><li> Ilgili genomik bölge ile etkileşime giren proteinlerin kantitatif belirlenmesi için, hücre kültürü (SILAC) enChIP (enChIP-SILAC) ile birleştirildi analizinde amino asitlerle stabil bir izotop işaretleme kullanımını dikkate. Not: SILAC analizi için Media ticari satıcılardan temin edilebilir. SILAC belirli etkileşimleri tespit güçlüdür. <ol><li> SILAC ağır ortam kültür hücreleri. Hücreler Cultu hazırlayınBir negatif kontrol olarak SILAC ışığı ortamı olarak verilmiştir. (Ağır veya hafif) her SILAC ortamı için en az 5 x 10 7 hücreleri kullanın. Etkili bir etiketleme için, her iki L-Lizin-2HCI, 13 ° C 6 ve L-Arginin-HCl, SILAC Ağır ortam içinde 13 ° C 6, 15 N 4 ekleyin. Not: en az beş hücre bölünmesi etiket protein için gereklidir. <ol><li> Örneğin, kararlı bir şekilde Lizin-2HCI artı L-Arginin-HCl (Işık ortamı) SILAC için DMEM ortam ve diyalize edilmiş fetal büyükbaş hayvan serumu, 37 ° C&#39;de ve% 5 CO2 seviyesinde 3xFLAG-dCas9 ve gRNA ifade kültürün insan fibrosarkoma HT1080 hücreleri ya da 13C 6 L-Lizin-2HCI artı 13 Cı 6 15 N 4 L-Arginin-HCl (Ağır ortam) 16 (Malzeme). Ortamın 500 ml hafif ya da ağır bir L-Lizin-2HCI ve L-Arginin-HCl, 50 mg ekleyin. </li><li> Hücreler üslü growt muhafaza böylece tripsinize ve replate hücreler konfluent hale gelmeden önceh. </li></ol></li></ol></li></ol> Formaldehit ile Hücreler 3. çapraz bağlanması <ol><li> Süspansiyon kültüründe hücreler, transfer 2 x 10 7 3xFLAG-TAL proteinler veya 3xFLAG-dCas9 artı gRNA eksprese eden hücreler ve 50 ml&#39;lik bir santrifüj tüpü içinde düzenli bir kültür ortamında 30 ml asılır. Daha fazla hücre kullanıldığında, orantılı reaktif hacimleri ve miktarlarını artırmak. SILAC deneyleri için, ağır ortam veya hafif ortamında (5 x 10 7 hücreleri her) kültüre hücrelerin karışımı ve 2 x 10 7 hücre ihtiva eden 5 tüp içine 1 x 10 8 hücre bölün. </li><li> Hücre süspansiyonu (nihai konsantrasyon% 1) 30 ml% 37 formaldehit 810 ul ilave edin ve 5 dakika boyunca 37 ° C&#39;de inkübe edin. Yapışık hücreler için, doğrudan kültür ortamında 30 ml% 37 formaldehit 810 ul ekleyerek kültür kapları içinde hücrelerin tespit. </li><li> 1,25 M glisin çözeltisi (nihai konsantrasyon 127 mM) 3.1 ml ekleyerek çapraz bağlanmasını durdurmak ve10 dakika boyunca oda sıcaklığında inkübe edilir. </li><li> Santrifüj (4 ° C &#39;de 5 dakika süre ile 300 x g) hücreleri toplamak. Dikkatle süpernatant dahil formaldehit atmak ve uygun bir atık şişe içinde saklayın. <ol><li> Yapışık hücreler, bir 50 ml tüp içinde, bir hücre kazıyıcı ile hasat hücreleri ayırmak ve bu aşamada tarif edildiği gibi hücreleri toplamak. </li><li> SILAC deneyleri için, ağır, orta veya hafif orta (5 x 10 7 hücre her) kültüre müstakil hücrelerin karışımı 2 x 10 7 hücre içeren 5 tüpler içine 1 x 10 8 hücrelerinin toplam bölmek ve bu tarif edilen hücreleri toplamak adım. </li></ol></li><li> Tüp başına iki kez PBS ile 30 ml hücre pelletini yıkayın. Dikkatle formaldehit içeren süpernatant atın ve kimyasal güvenlik yönergelerine göre formaldehit atık bottle.Handle uygun bir atık saklayın. Sabitlenen hücreler dondurulmuş ve -80 ° C&#39;de muhafaza edilebilir. </li></ol> Kromatin 4. Hazırlama (başına 2 x 107 hücreleri) <ol><li> Hücre lisis tamponu, 10 ml (10 mM Tris (pH 8.0), 1 mM EDTA,% 0.5 IGEPAL CA-630 ve 1 x proteaz inhibitörleri) sabit süspanse edilir ve 10 dakika süreyle buz üzerinde kuluçkalayın. </li><li> Santrifüj örneği (8 dakika boyunca 4 ° C&#39;de 830 x g) ve dikkatli bir supernatant atın. </li><li> Nükleer lizis tamponu 10 ml pelet Askıya (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 M NaCl,% 1 Triton X-100,% 0.5 sodyum deoksikolat,% 0.5 loroilsarkosin ve 1x proteaz inhibitörleri). 10 dakika süreyle buz üzerinde inkübe ve her 2-3 dakikada bir vorteks. </li><li> (830 × numuneyi santrifüj 8 dakika boyunca 4 ° C&#39;de g) ve dikkatli bir şekilde supernatant atın. </li><li> 10 ml PBS pelet yıkayın. Pelet (kromatin fraksiyonu) sıvı azot içinde hızlı soğutma, -80 ° C&#39;de saklanabilir. </li></ol> Kromatin 5. Sonication (2 x 10 7 hücre başına) <ol><li> 800 ul kromatin fraksiyonu Askıyadeğiştirilmiş lisis tamponu 3 (10 mM Tris (pH 8.0), 1 mM EDTA, 150 mM NaCI,% 0.1 sodyum deoksikolat,% 0.1 SDS ve 1x proteaz inhibitörleri) ve 1,5 ml tüp içine aktarılır. </li><li> Çıktı: (Malzeme) bir sonikatör kullanılarak kromatine ve aşağıdaki koşulları ses dalgalarına maruz 3; görev:% 100 (sürekli); ve saati: ücretsiz. 10 saniye boyunca, sonikasyon 10-18 döngü gerçekleştirmek ve 20 saniye süreyle buz üzerinde soğutuldu. Aşırı ısınmayı önlemek 2 dakika her 5-6 devir için buz üzerinde örnekleri inkübe. Köpük önlemek için, 0.5 cm tüpün tabanından sonikasyon probunun ucu konumunu tutun. </li><li> Santrifüj örneği (10 dakika boyunca 4 ° C&#39;de 16.000 x g) ve 1,5 ml tüp içine süpernatant aktarın. Sonike kromatin sıvı nitrojen içinde hızlı dondurma işleminin -80 ° C&#39;de saklanabilir. </li></ol> Kromatin Parçalanma 6. Değerlendirilmesi <ol><li> Distile su 85 ul parçalanmış kromatin 10 ul karıştırın. </li><li> 4 ul ekleve 5 M NaCI, 65 ° CO / N inkübe edin. </li><li> 10 mg / ml RNase A 1 ul ilave edin ve 45 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> 0.5 M EDTA (pH 8.0), 1 M Tris (pH 6.8) 4 ul, 20 mg 1 ul / ml proteinaz K 2 ul ekleyin ve daha sonra 1,5 saat süre ile 45 ° C&#39;de inkübe edin. </li><li> Örneğin SYBR Green I boyama boyalar içermeyen bir% 1 agaroz jeli içinde elektroforez ile numunenin ayrı ayrı 10 ul. </li><li> Parçalanmış kromatin uzunlukları dağılımını değerlendirmek için jel leke. 0.5-2 kbp (0,2-4 kbp&#39;lik aralığı) ortalama uzunluğu üreten şartlar tavsiye edilir. </li><li> Fenol-kloroform ekstraksiyonu ile veya bir DNA çıkarma kiti (Malzeme) kullanarak, kalan örneklerinin DNA saflaştınlır. Saflaştırılmış DNA enChIP analiz verimlerini (8.11) tahmin etmek için, giriş DNA olarak kullanılabilir. </li></ol> Antikorlar ile Dynabeads Konjuge 7. Hazırlama (2 x 10 7 hücre başına) <ol><li> Önİki 1,5 ml tüpler, normal fare IgG kullanılarak ön-temizleme için ve bir anti-FLAG Ab ile inkübasyon diğer pare. Her bir tüp, Protein G-konjuge manyetik boncuk 150 ul (Malzemeler bakınız) ekleyin. </li><li> Bir mıknatıs standına tüpler koyun ve 3 dakika bekleyin. Pipetleme süpernatant atın. </li><li> PBS% 0.01 Tween-20 (PBS-T) içeren 1 ml boncuk süspanse edin. Manyetik bir stand üzerinde tüpler koyun ve 2 dakika bekleyin. Pipetleme süpernatant atın ve sonra adımı tekrarlayın. </li><li> % 0.1 BSA içeren PBS-T 1 ml boncuk süspanse edin. </li><li> Normal fare IgG veya anti-FLAG Ab 15 ug ekleyin ve 4 ° CO / N dönmektedir. </li><li> Daha sonra, kısa bir süre aşağı Spin bir mıknatıs standına tüpler koyun ve 3 dakika bekleyin. Pipetleme süpernatant atın. </li><li> PBS-T 1 ml boncuk süspanse edin. Örnek birkaç kez ters çevirin ve kısaca aşağı doğru döndürün. Daha sonra pipetleme süpernatant atın, bir mıknatıs standında tüpler koyun ve 3 dakika bekleyin. Yıkama tekrarlayınPBS-T (üç yıkama adımları toplamı) ile iki kez daha. </li></ol> 8. Kromatin immünopresipitasyonu (2 x 10 7 hücre başına) <ol><li> Hacminin beşte biri% 5 Triton X-100 (% 1 Triton X-100 nihai konsantrasyon) ihtiva eden tadil edilmiş liziz tamponu 3 (yaklaşık 200 ul) ile 5.3 hazırlanan parçalanmış kromatin) (yaklaşık 800 ul) karıştırın. </li><li> Normal fare IgG&#39;si ile konjüge edilmiş Protein G-konjuge manyetik boncukları hazırlandı edildiği tüp kromatin solüsyonu ekleyin. 1 saat süre ile 4 ° C &#39;de döndürün. </li><li> Bir mıknatıs stand üzerinde tüp yerleştirin ve 3 dakika bekleyin. </li><li> Anti-FLAG Ab konjüge edilmiş Protein G-konjüge edilmiş manyetik boncukları hazırlandı edildiği tüp içine süpernatant aktarın. 4 ° CO / N at döndürün. </li><li> Bir mıknatıs stand üzerinde tüp yerleştirin ve 3 dakika bekleyin. Pipetleme süpernatant atın. </li><li> Düşük tuzlu tampon (20 mM Tris (pH 8.0), 2 mM EDTA, 150 mM NaCI, 1% Tri 1 ml boncuk Askıyatonluk X-100,% 0.1 SDS ve 1x proteaz inhibitörleri) ve 5 dakika boyunca 4 ° C&#39;de döner. Bir mıknatıs stand üzerinde tüp yerleştirin ve 3 dakika bekleyin. Pipetleme Süpernatantı atın ve yıkama adımı tekrarlayın. </li><li> Iki kez yüksek tuzlu tampon (20 mM Tris (pH 8.0), 2 mM EDTA, 500 mM NaCI,% 1 Triton X-100,% 0.1 SDS ve 1x proteaz inhibitörleri) ile boncuk yıkayın. </li><li> Iki kez LiCI tampon maddesi (10 mM Tris (pH 8.0), 1 mM EDTA, 250 mM LiCI,% 0.5 IGEPAL CA-630,% 0.5 sodyum deoksikolat ve 1 x proteaz inhibitörleri) ile boncuk yıkayın. </li><li> Bir kez TBS-IGEPAL CA-630 (50 mM Tris (pH 7.5), 150 mM NaCI,% 0.1 IGEPAL CA-630 ve 1 x proteaz inhibitörleri) ile boncuk yıkayın. </li><li> Elüsyon tamponu 200 ul boncuk Askıya (50 mM Tris (pH 7.5), 150 mM NaCl,% 0.1 IGEPAL CA-630, 1 x proteaz inhibitörleri ve 500 ug / ml 3 x FLAG peptidi), ve 37 ° C&#39;de inkübe edin 20 dakika. Bir mıknatıs stand üzerinde tüp yerleştirin ve 3 dakika bekleyin. Yeni 1.5 ml tüp içine süpernatant aktarın ve ELUT tekrariyon adım. </li><li> Fenol-kloroform ekstraksiyonu ile veya bir DNA ekstraksiyon kiti kullanılarak sıyırma sıvısının DNA küçük bir kısmı (örn, 5%) saflaştınlır (Malzeme). Saflaştırılmış DNA spesifik primer enChIP daha önce tarif edildiği gibi 14,16 Aşama 6.7&#39;de hazırlanan giriş DNA ile karşılaştırarak analiz verimlerini tahmin etmek için ayarlar, PCR için kullanılabilir. </li></ol> 9. SDS-PAGE, Boyama ve Kütle Spektrometrik Analizi <ol><li> 1 2-propanol ml 3M sodyum asetat (pH 5.2), 50 ul ve 20 mg / ml glikojen 5 ul eluat (400 ul) karıştırın. -20 ° CO / N de kromatin çöktürün. </li><li> Santrifüj örneği (30 dakika boyunca 4 ° C&#39;de 16.000 x g) ve süpernatan atılır. (10 dakika boyunca 4 ° C&#39;de 16.000 x g),% 70 etanol ve santrifüj yeniden 1 ml pelet durulayın. Pipetle tamamen süpernatant atın. </li><li> 2x numune tamponu (125 mM Tris (pH 6.8),% 10 2-merc 40 ul pelet Askıdaaptoethanol,% 4 SDS,% 10 sakroz ve% 0.004 bromofenol mavisi). 5 dakika vorteksleyin, tamamen pelet çözülür ve daha sonra proteinleri denatüre ve çapraz bağlantıyı ters döndürecek 30 dakika boyunca 100 ° C &#39;de inkübe etmek. </li><li> Boya çok altında 1 cm ulaşana kadar SDS-PAGE için jel üzerinde numunenin 40 ul çalıştırın. Not: Genellikle% 4-20 gradyan jelleri, ancak diğer% jeller kullanılabilmektedir. </li><li> Coomassie Brilliant Blue veya gümüş boyası ile jel Leke. </li><li> Beş adet (2 mm yükseklik) içine jel kesti. </li><li> Daha önce tarif edildiği gibi jel 13-16 sindirim ve kütle spektrometresi ile analiz gerçekleştirir. </li><li> 5 x 10 7 hücreleri, her (1 x 10 8 hücrelerin toplam) ile SILAC deneyleri için, 2x numune tampon maddesi 40 ul, ilk beş borunun (o kadar büyütmek için gerekli değildir) pelet askıya. </li></ol> RNA ve RNA-Seq Analizi 10. saflaştırılması <ol><li> EnChIP sonra RNA&#39;yı saflaştırmak için RNaz inhibitörü 5 U / ml ilave (seTampon çözelti tüm e malzemeler), değiştirilmiş lisis tamponu 3 ve elüsyon tamponu dışında olan RNaz İnhibitörü 40 U / ml ekleyin. </li><li> 5 M NaCI, 16 ul eluat (400 ul) karıştırın ve 2 saat süre ile 65 ° C&#39;de inkübe edin. </li><li> Örnek asit guanidinyum-fenol bazlı bir tepkime maddesi 1 ml (Malzemeler bakınız) ekleyin. Daha sonra 15 saniye vorteksleyin ve 5-15 dakika boyunca oda sıcaklığında inkübe edilir. 12.000 x g ve oda sıcaklığında 15 dakika boyunca santrifüjleyin. </li><li> Yeni 1,5 ml tüp süpernatantı aktarın ve p-bromoanisol 5 ul ilave edin. Daha sonra 15 saniye vorteksleyin ve 3-5 dakika için oda sıcaklığında inkübe edilir. 12.000 x g ve oda sıcaklığında 10 dakika boyunca santrifüjleyin. </li><li> Yeni 2 ml&#39;lik bir tüp içine süpernatant (yaklaşık 1 mi) aktarın ve 2-propanol 1 ml. Tüp ters çevirin ve 10 dakika boyunca oda sıcaklığında inkübe edilir. (Malzeme) bir RNA saflaştırma kiti bir kolona karışımı yerleştirin. 12.000 × 1 dakika boyunca santrifüj sütun g RT. </li><li> Yıkamabir RNA saflaştırma kiti RNA Yıkama Tamponu 400 ul (Malzeme) ile kolon. </li><li> DNase I (1 U / ul), 10 x DNase I Reaksiyon Tamponu ve 8 ul, DNase / RNase içermeyen su 3 ul, ve RNA Yıkama Tamponu 64 ul içinde 5 ul DNaz I kokteyl 80 ul ekle (karışım sütun bir RNA arıtma kiti (Malzeme)). 15 dakika boyunca 37 ° C&#39;de inkübe edin ve daha sonra numune 12.000 x g ve oda sıcaklığında 30 saniye boyunca santrifüj. </li><li> Iki kez bir RNA saflaştırma kiti RNA Ön Yıkama Tamponu 400 ul (Malzeme) ile sütun yıkayın. </li><li> DNase / RNase içermeyen su, 50 ul RNA Zehir. Elüt RNA, RNA sıralaması için kullanılabilir. </li></ol>

<ol>
	<li>Zhang, X. Y., Horz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+highly+purified+satellite+DNA+containing+chromatin+from+the+mouse.">Analysis of highly purified satellite DNA containing chromatin from the mouse.</a> Nucleic Acids Res. 10, 1481-1494 (1982).</li><li>Workman, J. L., Langmore, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoprotein+hybridization:+a+method+for+isolating+specific+genes+as+high+molecular+weight+chromatin.">Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin.</a> Biochemstry. 24, 7486-7497 (1985).</li><li>Boffa, L. C., Carpaneto, E. M., Allfrey, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+active+genes+containing+CAG+repeats+by+DNA+strand+invasion+by+a+peptide+nucleic+acid.">Isolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acid.</a> Proc. Natl. Acad. Sci. USA. 92, 1901-1905 (1995).</li><li>Jaskinskas, A., Hamkalo, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+initial+characterization+of+primate+satellite+chromatin.">Purification and initial characterization of primate satellite chromatin.</a> Chromosome Res. 7, 341-354 (1999).</li><li>Griesenbeck, J., Boeger, H., Strattan, J. S., Kornberg, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+specific+chromatin+segments+from+chromosomal+loci+in+yeast.">Affinity purification of specific chromatin segments from chromosomal loci in yeast.</a> Mol. Cell Biol. 23, 9275-9282 (2003).</li><li>Ghirlando, R., Felsenfeld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+studies+on+defined+heterochromatin+fragments+support+a+30-&#181;m+fiber+having+six+nucleosomes+per+turn.">Hydrodynamic studies on defined heterochromatin fragments support a 30-&#181;m fiber having six nucleosomes per turn.</a> J. Mol. Biol. 376, 1417-1425 (2008).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+proteins+associated+with+specific+genomic+loci.">Purification of proteins associated with specific genomic loci.</a> Cell. 136, 175-186 (2009).</li><li>Hoshino, A., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+chromatin+immunoprecipitation:+a+method+for+isolating+specific+genomic+regions.">Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions.</a> J. Biosci. Bioeng. 108, 446-449 (2009).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+idenification+of+insulator+components+by+insertional+chromatin+immunoprecipitation.">Direct idenification of insulator components by insertional chromatin immunoprecipitation.</a> PLoS One. 6, e26109 (2011).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+by+insertional+chromatin+immunoprecipitation+(iChIP)+with+a+second-generation+tagged+LexA+DNA-binding+domain.">Efficient isolation of specific genomic regions by insertional chromatin immunoprecipitation (iChIP) with a second-generation tagged LexA DNA-binding domain.</a> Adv. Biosci. Biotechnol. 3, 626-629 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locus-specific+biochemical+epigenetics+/+chromatin+biochemistry+by+insertional+chromatin+immunoprecipitation.">Locus-specific biochemical epigenetics / chromatin biochemistry by insertional chromatin immunoprecipitation.</a> ISRN Biochem. 2013, 913273 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+retaining+molecular+interactions+by+the+iChIP+system+using+recombinant+exogenous+DNA-binding+proteins.">Efficient isolation of specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous DNA-binding proteins.</a> BMC Mol. Biol. 15, 26 (2014).</li><li>Fujita, T., Kitaura, F., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+of+the+Thy28-MYH9+axis+in+B+cell-specific+expression+of+the+Pax5+gene+in+chicken+B+cells.">A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.</a> PLoS One. 10, (2015).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+proteins+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Biochem. Biophys. Res. Commun. 439, 132-136 (2013).</li><li>Fujita, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+telomere-associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP).">Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).</a> Sci. Rep. 3, 3171 (2013).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+proteins+associated+with+an+IFN&#947;-responsive+promoter+by+a+retroviral+expression+system+for+enChIP+using+CRISPR.">Identification of proteins associated with an IFN&#947;-responsive promoter by a retroviral expression system for enChIP using CRISPR.</a> PLoS One. 9, e103084 (2014).</li><li>Fujita, T., Yuno, M., Okuzaki, D., Ohki, R., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+non-coding+RNAs+associated+with+telomeres+using+a+combination+of+enChIP+and+RNA+sequencing.">Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing.</a> PLoS One. 10, e0123387 (2015).</li><li>Naito, Y., Hino, K., Bono, H., Ui-Tei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPRdirect:+software+for+designing+CRISPR/Cas+guide+RNA+with+reduced+off-target+sites.">CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites.</a> Bioinformatics. , (2014).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Esvelt, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal+Cas9+proteins+for+RNA-guided+gene+regulation+and+editing.">Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.</a> Nat. Methods. 10, 1116-1121 (2013).</li><li>Qi, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repurposing+CRISPR+as+an+RNA-guided+platform+for+sequence-specific+control+of+gene+expression.">Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.</a> Cell. 152, 1173-1183 (2013).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+binding+of+the+CRISPR+endonuclease+Cas9+in+mammalian+cells.">Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.</a> Nat. Biotechnol. 32, 670-676 (2014).</li><li>Kuscu, C., Arslan, S., Singh, R., Thorpe, J., Adli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+characteristics+of+off-target+sites+bound+by+the+Cas9+endonuclease.">Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.</a> Nat Biotechnol. 32, 677-683 (2014).</li><li>Cencic, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protospacer+adjacent+motif+(PAM)-distal+sequences+engage+CRISPR+Cas9+DNA+target+cleavage.">Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.</a> PLoS One. 9, 109213 (2014).</li><li>O'Green, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., Segal, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+analysis+of+Cas9+binding+specificity+using+ChIP-seq+and+targeted+sequence+capture.">A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.</a> Nucleic Acids Res. 43, 3389-3404 (2015).</li><li>de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shelterin:+the+protein+complex+that+shapes+and+safeguards+human+telomeres.">Shelterin: the protein complex that shapes and safeguards human telomeres.</a> Genes Dev. 19, 2100-2110 (2005).</li></ol>

The authors have filed a patent on enChIP (Patent name: 'Method for isolating specific genomic regions using DNA-binding molecules recognizing endogenous DNA sequences'; Patent number: PCT/JP2013/74107). H.F. is a member of the Advisory Board of Addgene.

Here, we describe the purification of specific genomic regions using engineered DNA-binding molecules such as the CRISPR system and TAL proteins, and the identification of proteins and RNAs bound to these genomic regions. Binding of engineered DNA-binding molecules to the genome may affect chromatin structure, including nucleosome positioning, and may abrogate genomic functions, as described in CRISPR interference experiments21. To avoid these potential aberrant effects, we propose specific guidelines for choosing target genomic regions. First, to avoid potential inhibition of the recruitment of RNA polymerases and transcription factors, as well as disruption of nucleosome positioning around the transcription start site, the target regions for analyses of promoter regions should be several hundred base pairs upstream of (5' to) the transcription start site. By contrast, when analyzing genomic regions with distinct boundaries, such as enhancers and silencers, genomic regions that are directly juxtaposed to these regions can be targeted because it is less likely that the binding of engineered DNA-binding molecules will affect their functions. Furthermore, it is best to avoid using target regions that are conserved among different species, because important DNA-binding molecules often bind to evolutionarily conserved regions and inhibition of their binding might disrupt the functions of the target genomic regions. In this regard, it is always necessary to check that the function of the target genomic region is maintained in the established cells used for enChIP analyses. Because multiple gRNAs can be tested easily and it is tedious and expensive to generate multiple versions of TAL or zinc-finger proteins recognizing different target genomic regions, enChIP using CRISPR is more advantageous than enChIP using other proteins.
It has been shown that dCas9 binds to off-target sites although affinity to those sites might be weaker than that to the target sites22-25. There are several ways to manage contamination of molecules bound to those off-target sites. First, the use of several, at least two, different gRNAs would be recommended. Those molecules commonly observed in enChIP using distinct gRNAs would be true positives. Second, comparison of different conditions for enChIP would be effective in cancelling contamination of non-specific molecules and molecules bound to off-target sites. Examples of those comparison sets would be (i) stimulation (-) and (+), or (ii) different cell types such as T cells vs. B cells. Finally, quantitative analysis of binding of candidate molecules should be performed to confirm their specific binding to the target sites. It is preferable to prepare cells expressing only dCas9 but not gRNA as a negative control.
Using enChIP analyses, we were able to successfully identify a number of known and novel molecules interacting with specific genomic regions (Tables 1-3)14-17. However, this technique failed to detect some other known proteins interacting with these regions. For example, STAT1 reportedly associates with the IRF-1 promoter upon IFN&#947; stimulation8, but our enChIP-SILAC analysis did not detect STAT1 as a protein induced to interact with this genomic region16. In addition, in the enChIP-MS analysis of telomeres, we did not detect shelterin proteins consisting of TRF-1 and TRF-215, which have been shown to interact with telomeres26. There are a few potential reasons for these discrepancies. First, the stoichiometry of binding of Stat1 to the IRF-1 promoter might be very low. It is reasonable that enChIP-MS, including enChIP-SILAC, detects proteins that are more abundantly associated with target genomic regions; hence, the analysis of more cells might be necessary to detect these proteins. Increases in the sensitivities of MS instruments would also contribute to the efficient detection of proteins with low stoichiometric binding. Second, some proteins, possibly including Stat1 and shelterins, might be difficult targets for MS analyses. Third, in our analysis of telomere-binding proteins15, the 3&#215;FLAG-TAL proteins recognizing telomeres (3&#215;FN-Tel-TAL) might have blocked the binding of shelterins to telomeres in a competitive fashion.
In contrast to the relative difficulty of detecting transcription factors binding to specific genomic regions using enChIP, we successfully identified epigenetic regulators such as histone modification enzymes using enChIP analyses. The success of this technique may be due to the fact that epigenetic regulators bind to a broad range of genomic regions; hence, more proteins per genomic region are available for MS. Because epigenetic regulators are increasingly recognized as important targets for drugs against intractable diseases such as cancer, enChIP would be a useful tool for the identification of epigenetic drug targets.

Özel ilgi genomik bölgeleri ile ilişkili moleküllerin belirlenmesi gibi transkripsiyon ve epigenetik düzenleme gibi genomik fonksiyonların düzenlenmesinde mekanizmalarını anlamak için gereklidir. Çeşitli teknikler özel genomik bölgelere 1-7 biyokimyasal analizi için geliştirilmiş olmasına rağmen, bunlar, çünkü bu tür sınırlı bir uygulama (örneğin, sadece tekrarlar yüksek kopya sayısı lokusları veya lokus için) ve bunların iç sorunlar bu aşamada yaygın olarak kullanılmamaktadır çok fazla zaman ve çaba gerektiriyordu. Kolayca belirli genomik bölgelerin biyokimyasal analizini gerçekleştirmek amacıyla, biz 14-17 iki lokus spesifik kromatin immunoprecipitation (ChIP) teknolojileri, yani sokma ChIP (iChip) 8-13 ve mühendislik DNA bağlayıcı molekül aracılı ChIP (enChIP) geliştirdik . IChip olarak, ilgi konusu bir lokus gibi Lex gibi dışsal bir DNA-bağlama proteini sokulması tanıma sekansları tarafından etiketlenmişA. lokusu sonra etiketli bir DNA-bağlama proteini kullanılarak afinite saflaştırma ile izole edilmektedir. EnChIP olarak, örneğin çinko parmak proteinleri, transkripsiyon aktivatörü gibi (TAL) protein gibi DNA-bağlama molekülleri, yapay ve kümelenmiş düzenli aralıklarla yerleştirilmişlerdir kısa palindromik tekrarları (CRISPR) kompleksleri, (Şekil 1) ilgi konusu bir lokusu etiketlemek için kullanılır. Daha sonra, genomik bölge ile işaretlenmiş DNA-bağlama moleküllerinin afinite saflaştırma ile izole edilmektedir. IChip fazla enChIP avantajlarından biri bir ekzojen DNA-bağlama proteini tanıma sekansları sokulması gerekli değildir olmasıdır. Cas9 (dCas9) içindeki bir katalitik aktif olmayan biçimini kapsayan CRISPR kompleksleri ve bir kılavuz RNA (gRNA) ile lokusların Hedefleme TAL ve çinko-parmak proteinleri kullanılarak iChip veya enChIP bu bölgelerin hedeflemeyi çok daha kolaydır. Burada, kütle spektrometrisi ve RNA dizi analizi ile kombine enChIP için adım adım protokol açıklar (RNA-Seq) lokusa Associa tespit etmekted proteinleri ve RNA&#39;lar sırasıyla.

<table border="0" cellpadding="0" cellspacing="0" width="964">
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			<td height="38" style="height:38px;width:239px;">gBlock synthesis service</td>
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			<td height="21" style="height:21px;width:239px;">pSIR-neo</td>
			<td>Addgene</td>
			<td style="width:175px;">51128</td>
			<td style="width:353px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">pSIR-GFP</td>
			<td>Addgene</td>
			<td style="width:175px;">51134</td>
			<td style="width:353px;"></td>
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			<td height="21" style="height:21px;width:239px;">pSIR-DsRed-Express2</td>
			<td>Addgene</td>
			<td style="width:175px;">51135</td>
			<td style="width:353px;"></td>
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			<td height="21" style="height:21px;width:239px;">pSIR-hCD2</td>
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			<td style="width:175px;">51143</td>
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			<td style="width:175px;">47948</td>
			<td style="width:353px;"></td>
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			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-puro</td>
			<td>Addgene</td>
			<td style="width:175px;">51240</td>
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			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-IG</td>
			<td>Addgene</td>
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			<td height="20" style="height:20px;width:239px;">3&#215;FLAG-dCas9/pMXs-neo</td>
			<td>Addgene</td>
			<td style="width:175px;">51260</td>
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			<td height="21" style="height:21px;width:239px;">anti-FLAG M2 Ab</td>
			<td style="width:199px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:239px;">FITC-conjugated anti-FLAG M2</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:175px;">F4049</td>
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			<td>Other medium can be purchased from Life Technologies</td>
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			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">89986</td>
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			<td>for Light medium</td>
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			<td style="width:175px;">89988</td>
			<td>For Heavy medium</td>
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			<td style="width:175px;">89990</td>
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			<td style="width:199px;">Zymo Research</td>
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			<td height="21" style="height:21px;width:239px;">Dynabeads-Protein G</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:175px;">DB10004</td>
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			<td style="width:175px;">N2611</td>
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			<td height="21" style="height:21px;width:239px;">Isogen II</td>
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			<td style="width:199px;">Zymo Research</td>
			<td style="width:175px;">R2050</td>
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			<td height="21" style="height:21px;width:239px;">LTQ Orbitrap Velos</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">nanoLC</td>
			<td style="width:199px;">Advance, Michrom Bioresources</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
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			<td height="21" style="height:21px;width:239px;">HTC-PAL autosampler</td>
			<td style="width:199px;">CTC Analytics</td>
			<td style="width:175px;"></td>
			<td>a component of a nanoLC-MS/MS system for MS analysis</td>
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	</tbody>
</table>

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.

Genel olarak, bir hedef genomik bölgelerinden% 1-30. EnChIP kullanılarak arıtılmış, 3 enChIP verimlerini gösteren temsili verileri içermektedir Şekil edilebilir telomerlerin ve interferon (IFN), düzenleyici faktörü-1 (IRF-1) gen promotörü hedef analizleri. Tipik sonuçlar, Tablo 1, örnek olarak enChIP-SILAC, Tablo tarafından tanımlanan bir IFNy özgü bir şekilde IRF-1 promotör ile ilişkili proteinler 2 listeler ile kütle spektroskopisi enChIP tarafından tanımlanan telomer bağlayıcı proteinler (enChIP-MS) ve Tablo 3 enChIP-RNA-Sek tarafından belirlenen telomerlerin ilişkili RNA&#39;lar. <img alt="figür 1" src="/files/ftp_upload/53478/53478fig1.jpg" /> EnChIP 1. Genel Bakış Şekil. enChIP kullanılarak CRISPR (A, B) ve TAL (C, D, E </strong&gt;) gösterilir. Ilgi odağı bir böyle bir TAL protein veya Cas9 (DCAS) bir katalitik inaktif formda oluşan CRISPR sistemi olarak tasarlanmış DNA bağlama molekülleri ile etiketlendi ve (gRNA) RNA rehberlik edilir. Moleküler etkileşimleri formaldehit ya da başka çapraz bağlayıcı maddelerin, gerektiğinde ile sabitlenir. Daha sonra, sabit kromatin sonication veya enzimatik sindirimi ile parçalanır. Etiketli genomik bölge afinite saflaştırma ile saflaştırılır. Son olarak, çapraz tersidir ve etkileşim molekülleri (genomik bölgelerde, RNA&#39;lar ve proteinler) yeni nesil sıralama ve kütle spektrometresi kullanılarak tanımlanır. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53478/53478fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/53478/53478fig2.jpg" /> GBlock Şekil 2. Sıra. U6 organizatörü (siyah), G nükleotid before kılavuz dizisi (mavi), kılavuz dizisi (kırmızı) (gRNA boşluk), (yeşil) iskele dizisi ve (turuncu) terminatör dizisi gösterilir. <img alt="Şekil 3," src="/files/ftp_upload/53478/53478fig3.jpg" /> Temsili enChIP analizleri 3. Bir Getirisi Şekil. Hedefe IRF-1 odağı ve hedef olmayan Sox2 lokus için (A) Yüzde girişi. Hedef telomer ve hedef olmayan γ-uydular için (B) Yüzde girişleri. Rakamlar uyarlanmış ve önceki yayınlardan 15,16 den modifiye edilmiştir. <table fo:keep-together.within-page="1"><tbody><tr><td> Kategoriler </td><td> Proteinler </td></tr><tr><td> Transkripsiyon </td><td> DDX1, PARP1&#39;i, CKAP4, Pescadillo homologu, PURβ, aktive edilmiş RNA polimeraz II transkripsiyonel aktivatör p15, BTF3, Myb bağlayıcıProtein 1A </td></tr><tr><td> Histon deasetilasyonu, corepressor bileşenleri </td><td> RBBP4, PA2G4, TBL3 </td></tr><tr><td> Asetiltransferaz </td><td> Protein arginin, N-metiltransferaz 1 </td></tr><tr><td> DNA topoizomeraz </td><td> DNA topoizomeraz 2α </td></tr><tr><td> Histonlar </td><td> Histon H2A.Z histon H3.2 </td></tr></tbody></table> Tablo 1:. EnChIP-SILAC tarafından tanımlanan bir IFNy-spesifik bir şekilde insan IRF-1 promotör bölgesi ile ilişkili proteinlerin örnekleri tablo önceki yayın 16 adapte edilmiştir. <table fo:keep-together.within-page="1"><tbody><tr><td> Kategoriler </td><td> Proteinler </td></tr><tr><td> Memeli telomer-bağlayıcı proteinler </td><td> PML, RPA, CDK1, PARP1&#39;i, PCBP1 </td></tr><tr><td> Telomer-biMayada nding proteinler ya da diğer organizmalar </td><td> IMP4 </td></tr><tr><td> Ile etkileşim Proteinler telomer bağlayıcı proteinleri [ilişkili telomer bağlayıcı protein] </td><td> DNA polimeraz α (POLA1) [Cdc13p], ARMC6 [TRF2] CTBP1 [FoxP2-POT1], exportin-5 [TERT] GNL3L [TRF1], exportin-1 [TERT], 14-3-3 [TERT] </td></tr><tr><td> Heterokromatindir için lokalize Proteinler </td><td> BEND3 </td></tr><tr><td> Proteinler epigenetik işaretleri düzenleyen </td><td> KDM5C </td></tr><tr><td> Kimin mutasyonlar telomer fonksiyonunu etkileyen Proteinler </td><td> DNA polimeraz α (POLA1), HAT1, Nup133, CDK7, DPOE1, PRDX1, TYSY, Glutamat-sistein ligazı, glutaredoksin, SMRC1 </td></tr></tbody></table> Tablo 2:. EnChIP-MS yolu ile teşhis fare telomerlerin ilişkili proteinlerin örnekleri, Tablo adaptö olmuşturBir önceki yayın 15 den d. <table fo:keep-together.within-page="1"><tbody><tr><td> Kategoriler </td><td> RNA&#39;lar </td></tr><tr><td> Telomeraz bileşenleri </td><td> Terc, Rmrp </td></tr><tr><td> Telomer RNA&#39;lar </td><td> Terras </td></tr><tr><td> scaRNAs </td><td> Scarna6, Scarna10, Scarna13, Scarna2 </td></tr><tr><td> H / ACA snoRNAs </td><td> Snora23, Snora74a, Snora73b, Snora73a </td></tr><tr><td> C / D snoRNAs </td><td> Snord17, Snord15a, Snord118 </td></tr><tr><td> lncRNA </td><td> Neat1 </td></tr></tbody></table> Tablo 3:. EnChIP-RNA-Seq tablo önceki yayın 17 den adapte edilmiştir tarafından belirlenen fare telomerlerin ile ilişkili RNA&#39;lar örnekleri.

Watch this Scientific Journal Video about Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP at JoVE.com

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

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

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.

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

isolation-specific-genomic-regions-identification-associated

Nanyang Technological University

Research

JoVE Journal

Biology

10.4K Görüntüleme.  Osaka University. 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.

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

Isolation of Genomic DNA from Mouse Tails

Fare Tails Genomik DNA izolasyonu

Biyoloji

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Spesifik endo-ve exoglycosidases kullanarak protein glikozilasyon tanımlanması ve karakterizasyonu

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Floresans Aktif Hücre Ayırma (FACS) Taze Dokular Normal ve Kanser ilişkili Fibroblastlar İzolasyonu

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Of Utility Sahne özgü Orta-geç<em&gt; Drosophila</em&gt; Folikül İzolasyon

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized near the mitochondria. Support for this possibility is derived from recent studies, in which many mRNAs encoding mitochondrial proteins were shown to be localized to the mitochondria vicinity. Together with earlier demonstrations of ribosomes&#8217; association with the outer membrane, these results suggest a localized translation process. Such localized translation may improve import efficiency, provide unique regulation sites and minimize cases of ectopic expression. Diverse methods have been used to characterize the factors and elements that mediate localized translation. Standard among these is subcellular fractionation by differential centrifugation. This protocol has the advantage of isolation of mRNAs, ribosomes and proteins in a single procedure. These can then be characterized by various molecular and biochemical methods. Furthermore, transcriptomics and proteomics methods can be applied to the resulting material, thereby allow genome-wide insights. The utilization of yeast as a model organism for such studies has the advantages of speed, costs and simplicity. Furthermore, the advanced genetic tools and available deletion strains facilitate verification of candidate factors.

Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions&#160;in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria.

Lokalize Çeviri Mekanizmaları Eğitim için Maya Mitokondri ile mRNAlarının Associated İzolasyonu

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Antik Biyolojik Topluluklar barındırmıştır İç Araştırma Dondurulmuş Çekirdek dış bölümünden Eksojen Malzeme Kaldırma

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

Bitki organizatörü analizleri: Kimlik ve kök nodül belirli organizatörü ortak fasulye içinde karakterizasyonu

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

Vibratom Kesitleme ile Fare Renal Pelvisin Canlı, Sağlam Kalp Pili Bölgelerini İzolasyon ve Görüntüleme

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...