Yazarlar Singapur A * STAR tarafından desteklenmektedir. Buna ek olarak, MJF Bilim Bursu ve Lee Kuan Yew Doktora Sonrası Araştırma Bursu kadınlar için bir A * STAR Ulusal Bilim Bursu, bir L&#39;Oreal tarafından desteklenmektedir. YR NIH KODLAMASI hibe (R01 HG004456-01 ve R01 HG003521-01) tarafından desteklenmektedir. Yazarlar ayrıca video düzenleme ve için Bayan Michelle Teo için Bayan Siti Rahim sahneleri filme özellikle Sayın Kelvin Issey, Bay Chang Kai Xiang ve Sayın Sherwin Gan 8 Piksel Productions, Singapur, videografisi ekibi kabul voice-over.

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Çıkar çatışması ilan etti.

Chia-PET transkripsiyon regülasyonu uzun menzilli etkileşimleri tanımlamak için geliştirilmiş bir yöntemdir. Bir Chia-PET kütüphane kalitesini belirleyen önemli faktörlerden biri ChIP malzeme kalitesidir. Video gösterilen protokol hücre çapraz-bağlamak için EGS ve formaldehit kullanımını içerir. Uzun ayırıcı kolu taşıyan ikinci bir çapraz bağlama reaktifi ile birlikte formaldehit tek başına kullanımı 3,11,15 formaldehit bağlı olamazdı proteinlerin bağlanmasında yardımcı olabilir. Biz sağlam bağlayıcı siteleri ve uzun menzilli etkileşimleri 9 göstermiştir bu yöntem ile kütüphaneler inşa ettik. Ancak, çapraz bağlama ve ChIP koşulları ilgi her faktör için optimize edilmeli ve çapraz bağlama çok sonication tarafından parçalanma zorluklara neden olacak gibi değil aşırı kroslink önemlidir ve muhtemelen sahte kromatin etkileşimleri neden olabilir . Kromatin etkileşimleriChia-PET tarafından tanımlanan tür floresan in-situ hibridizasyon 4 gibi farklı bir yöntem ile valide edilmelidir. Biz kromatin malzeme 100 ng asgari öneririz. Biz kromatin malzeme 50 ng kaliteli kütüphaneler inşa ederken, biz başlangıç ​​materyali büyük miktarlarda izin verdiğini gözlemledim az 16 PCR döngüleri, böylece minimize amplikonlarının ve her kütüphane artıklık ile Chia-PET kütüphanelerinin yapımı. Bu düşük artıklık böylece sıralama daha az şeritli daha kapsamlı bir kromatin etkileşim haritası sağlayan, yüksek benzersiz eşlenen etiketleri ve ayrıca kullanılabilir veri oranı yüksek korelasyon. Her bir tüp içinde boncuk nihai paketlenmiş hacim sırasıyla manyetik ve Sepharose boncuklar için 50 ul ve 100 ul olmalıdır. Dolu boncuk hacmi belirtilenden daha az ise, DNA taşıyan boncuk kaybını en aza indirmek için benzer önceden temizlenmiş boş manyetik ya Sepharose boncuklar ile minimum dolu ses getirmeksonraki adımlarda s. Sawed-off ipucu ya da çok-çekirdekli kriteri Sepharose boncuklar pipetleme için kullanılmalıdır. Aşağıdaki değişiklikler önceden yayınlanmış Chia-PET protokolü 5 takiben dahil edildi. Öncelikle, manyetik G boncuk yıkama sırasında numune kaybını minimize etmek için kullanılmıştır. Ayrıca, kendi kendine bağlanan yarım bağlayıcılar ve / veya adaptörler amplikonlarının olacak 100 bp ve 138 bp yaklaşık boyutları ile non-spesifik bantları belirlendi. Bu yüzden, PCR amplifikasyon sırasında spesifik olmayan şeritler en aza indirmek için biyotinile yarı-bağlayıcılara ve 454 GS20 adaptörleri konsantrasyonu azalır. Yakınlığı ligasyonu hacmi sonraki saflaştırma adımları sırasında numune kaybını en aza indirmek ve aynı zamanda reaktif maliyetlerinde tasarruf için 50 ml 10 ml düşürülmüştür. Biz de streptavidin boncuklar üzerinde Chia-PET DNA maksimal yakalama sağlamak için boncuk Chia-PET DNA sabitlemek için inkübasyon süresi arttı. Yakınlığı ligasyonu adım sırasında, kimerik ligasyonu that kaçınılmaz bir spesifik olmayan ve rasgele bir şekilde üretilir vivo kromatin etkileşimleri gerçek temsil etmemektedir. Bu nedenle, herhangi bir Chia-PET deneme veri kalitesini değerlendirmek için, kimerizm oranı belirli nükleotid barkod TAAG ve ATGT 5 ile iki farklı yarı linkers kullanımı tahmin edilmektedir. Yüksek verimli sekanslama sonra, Chia-PET dizileri ilk 8 ayırt edilebilen belirli Ligasyon ürünleri ve non-spesifik ürünlerden elde edilen ligasyon bağlayıcının barkod kompozisyon ve sekansları için analiz edilir. Bilinen kimeralar yüzdesi (yani heterodimerler AB linkers) bizim in-house MCF-7 RNA Polimeraz II Chia-PET kütüphaneler az% 15 sunuyoruz. Chia-PET dizileri sonradan olmak üzere iki kategoride, kendinden ligasyonu PET ve inter-ligasyon PET ayrılır. Arası ligasyon PET ı elde edilir kendinden-ligasyon PET kromatin fragmanlarının kendi Sirkülasyon ligasyon elde edilmiştiriki farklı DNA parçaları arasında nter-ligasyonu. İkincisi daha sonra aynı kromozom (intrachromosomal arası ligasyonu PET) veya iki etiketi iki farklı kromozom (interchromosomal arası ligasyonu PET) eşlenen her etiketi genomik mesafeye göre üç farklı kategoride alt grupları oluşturmaktadır. Biz farklı kategorilerde 8 dışarı sıralamak için bir Chia-PET aracı yazılım paketi geliştirdik. Bu kütüphane olan DNA fragmanları üzerinde bağlı olacaktır. Genellikle, küçük ChIP parçaları daha yüksek bir çözünürlüğe vermek ve 4 kb hakkında bu RNA Polimeraz II Chia-PET kütüphaneler için kesecek. Buna ek olarak, gerçek kromatin etkileşimleri bir etkileşim kümedeki arası ligasyonu PET sayısını sayarak rastgele gürültü ayırt edilebilir, diğer bir deyişle, yüksek PET sayısı bir küme gerçek kromatin etkileşimi 8 olma ihtimali daha yüksek olduğu söyleniyor . Bir yalancı pozitif filtrelemek içinrastgele tesadüflerle arası ligasyonu PET oluşabilir zenginleştirilmiş çapa yükselecek, bir istatistiksel analiz çerçevesi de iki ankraj 8 arasındaki inter-ligasyon PET rasgele oluşumu için hesap için formüle edilmiştir. Sonuç olarak, Chia-PET tekniği küresel ölçekte kromatin etkileşim ağları haritalama sağlar. Chia-PET ChIP uygulanması kütüphanesi karmaşıklığı ve arka plan gürültüsünün azalmasını sağlar. Buna ek olarak, belirli ChIP transkripsiyon faktörleri 5 ile bağlantılı spesifik kromatin etkileşimlerin inceleme sağlayan, kromatin etkileşimleri için özgüllüğü ekler.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog Numarası </td><td> Yorumlar </td></tr><tr><td> % 4-20 gradyanı TBE jel </td><td> Invitrogen </td><td> EC6225BOX </td><td> B, 6.3 Adım </td></tr><tr><td> PEG ile 5 x T4 DNA Ligaz Tamponu </td><td> Invitrogen </td><td> 46300018 </td><td> B aşaması, 2.2 </td></tr><tr><td> % 6 TBE jeli </td><td> Invitrogen </td><td> EC6263BOX </td><td> B, 6.8 Adım </td></tr><tr><td> Agilent DNA 100 Testi </td><td> Agilent Technologies </td><td> 5067-1504 </td><td rowspan="3"> B aşaması, 7.1 </td></tr><tr><td> Agilent Yüksek Hassasiyet DNA Testi </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> (Spin-X) Tüp Filtreler santrifüjleyin </td><td> Corning &lt;/ Td&gt; <td> CLS8160 </td><td> Adım B, 3.7 ve 6.9 </td></tr><tr><td> Koyu Okuyucu transilluminator </td><td> Clare Kimya Araştırma </td><td> DR46B </td><td> B, 6.8 Adım </td></tr><tr><td> Dijital Sonifier Hücre bölücü </td><td> Branson </td><td> 450D-0101063591 </td><td> A, 4.3 Adım </td></tr><tr><td> DynaMag-2 mıknatıs (Manyetik Parçacık Yoğunlaştırıcı) </td><td> Invitrogen </td><td> 123-21D </td><td> Adımları yıkama tüm manyetik boncuk, A, 7.5.1 Adım Adım B </td></tr><tr><td> DynaMag-15 Mıknatıs (Manyetik Parçacık Yoğunlaştırıcı) </td><td> Invitrogen </td><td> 123.01D </td><td> A, 5 ve 6 Adım </td></tr><tr><td> DynaMag-PCR (Manyetik Parçacık Yoğunlaştırıcı) </td><td> Invitrogen </td><td> 49-2025 </td><td> B, 6.5 Adım </td></tr><tr><td> Escherichia coli DNA polimeraz I </td><td> NEB </td><td> M0209 </td><td> B, 5.6 Adım </td></tr><tr><td> GlycoBlue </td><td> Ambion </td><td> AM9516 </td&gt; <td> A, 7.5.1 Adım B, 4.3 ve 6.5 Adım </td></tr><tr><td> Illumina CBOT Küme Üretim Sistemi </td><td> Illumina </td><td> SY-301-2002 </td><td> B aşaması, 7.4 </td></tr><tr><td> Illumina Genom Analyzer IIx </td><td> Illumina </td><td> SY-301-1301 </td><td> B aşaması, 7.5 </td></tr><tr><td> Illumina PE astar </td><td> Illumina </td><td> PE-102-1004 </td><td> Adım B, 5.4 (gerekli ise) </td></tr><tr><td> Intelli-Mikser </td><td> Palico Biyoteknoloji </td><td> RM-2L </td><td> , Rotasyon ile herhangi inkübasyonlar B Adım </td></tr><tr><td> LightCycler 480 Real-Time PCR Sistemi </td><td> Roche </td><td> 04 640 268 001 </td><td> A, Adım B 7.5.3, 7.3 Adım </td></tr><tr><td> LightCycler480 DNA SYBR Green I MasterMix </td><td> Roche </td><td> 03 752 186 001 </td><td> Adım B, 7.5.3 </td></tr><tr><td> M-280 Streptavidin Dynabeads </td><td> Invitrogen </td><td> 11206D </td><td> B, 5.2 Adım </td></tr> &lt;tr&gt; <td> Manyetik (Dynabeads) Protein G </td><td> Invitrogen </td><td> 100.03D </td><td> A, 5.1.1 ve 5.2.1 Adım </td></tr><tr><td> Yüksek Yoğunluk (2ml) MaXtract </td><td> Qiagen </td><td> 129056 </td><td> A, Adım 7.5.1 </td></tr><tr><td> Yüksek yoğunluklu (50ml) MaXtract </td><td> Qiagen </td><td> 129073 </td><td> B aşaması, 4.2 </td></tr><tr><td> MmeI </td><td> NEB </td><td> R0637 </td><td> R0637 </td></tr><tr><td> RNaz ONE Ribonükleaz </td><td> Promega </td><td> M426C </td><td> B aşaması, 4.8 </td></tr><tr><td> RNA polimeraz II (8WG16) monoklonal antikor </td><td> Covance </td><td> MMS-126R </td><td> A, Adım 5.2.4 </td></tr><tr><td> Oak Ridge Santrifüj Tüpleri (Polipropilen) </td><td> Nalgene </td><td> 3119-0050 </td><td> A, 3.1 Adım </td></tr><tr><td> Oak Ridge Santrifüj Tüpleri (Teflon FEP) </td><td> Nalgene </td><td> 3114-0050 </td><td> B aşaması, 4.3 </td></tr><tr><td> Phusiyon High Fidelity Master Mix </td><td> Finnzymes </td><td> F-531 </td><td> B, 6 Adım </td></tr><tr><td> PicoGreen (Quant-iT) dsDNA Reaktif </td><td> Invitrogen </td><td> P11495 </td><td> A, 7.5.2 Adım </td></tr><tr><td> Polistiren Yuvarlak Dip Tüp </td><td> BD Biosciences </td><td> 352057 </td><td> A, 4.1 Adım </td></tr><tr><td> Proteaz İnhibitör Kokteyl tabletler (tam, EDTA-ücretsiz) </td><td> Roche </td><td> 11873580001 </td><td> A, 1.6 yılından Adım </td></tr><tr><td> Proteinaz K Çözüm (20mg/ml) </td><td> Fermentas </td><td> E00491 </td><td> 7.5.1, 4.4 Adım A B aşaması, 4.1 </td></tr><tr><td> T4 DNA Ligaz </td><td> Fermentas </td><td> EL0013 </td><td> Adım B, 2.2, 3.9 ve 5.4 </td></tr><tr><td> T4 DNA Ligaz Tamponu (NEB) </td><td> NEB </td><td> B0202S </td><td> Adım B, 3.3 ve 3.9 </td></tr><tr><td> T4 DNA polimeraz </td><td> Promega </td><td> M4215 </td><td>B aşaması, 1.2 </td></tr><tr><td> T4 DNA polinükleotid kinaz </td><td> NEB </td><td> M0201 </td><td> B, 3.3 Adım </td></tr><tr><td> TruSeq SBS Takımı v5-GA </td><td> Illumina </td><td> FC-104-5001 </td><td> Illumina Genom Analyzer IIx sistemi için Regents </td></tr><tr><td> TruSeq PE Küme Takımı v2-CBOT-GA </td><td> Illumina </td><td> PE-300-2001 </td><td> Illumina CBOT Küme Üretim Sistemi ile kullanım olmak </td></tr><tr><td> SYBR Green I </td><td> Invitrogen </td><td> S-7585 </td><td> Adım B, 6.3 ve 6.8 </td></tr><tr><td> XCell SureLock Mini Hücre Elektroforez Sistemi </td><td> Invitrogen </td><td> EI0001 </td><td> Adım B, 6.3 ve 6.8 </td></tr></tbody></table><table border="1"><tbody><tr><td> Isim </td><td colspan="2"> Sıra </td><td> Yorumlar </td></tr><tr><td rowspan="2"> Biyotinile yarı-bağlayıcı A (200ng/μl) </td><td> Üst </td><td> 5 &#39;GG CCG CGA / iBiodT / ATC TTA TTK AAC 3&#39; </td><td> 250 nmole ölçekte HPLC Saflaştırılmış İç Biotin dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA TAA GAT ATC GC 3&#39; </td><td> 250 nmole ölçekte HPLC Saflaştırılmış </td></tr><tr><td rowspan="2"> Biotinlenmiş yarım linkers B (200ng/μl) </td><td> Üst </td><td> 5 &#39;GGC CGC GA / iBiodT / ATA CAT TTK AAC 3&#39; </td><td> 250 nmole ölçekte HPLC Saflaştırılmış İç Biotin dT (9) </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA ATG TAT ATC GC 3&#39; </td><td> 250 nmole ölçekte HPLC Saflaştırılmış </td></tr><tr><td rowspan="2"> Sigara biotinlenmiş yarım linkers (200ng/μl) </td><td> Üst </td><td> 5 &#39;GGC CGC GAT ATC GGA TCC AAC 3&#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td> Bot </td><td> 5 &#39;GTT GGA TCC GAT ATC GC 3&#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td rowspan="2"> GS20 Adaptör A (200ng/μl) </td><td> Üst </td><td> 5 &#39;CCA TCT CAT CCC TGC GTG TTK CATCTG TTC SKK CCC TGT CTC AGN N 3 &#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td> Bot </td><td> 5&#39;CTG AGA CAG GGA GGG AAC AGA TGG GAC ACG CAG GGA TGA GAT GG 3 &#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td rowspan="2"> GS20 Adaptör B (200ng/μl) </td><td> Üst </td><td> 5 &#39;CTG AGA CAC GKRY ACA GGG GAT AGG CAA GGC ACA CAG GGG ATA GG 3&#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td> Bot </td><td> 5 &#39;CCT ATC CCC TGT GTG SKK TGC CTA TTK SKK GTT GCG TGT CTC AGN N 3&#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td rowspan="2"> Illumina-NN adaptörleri </td><td> Üst </td><td> 5 &#39;ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC 3&#39; </td><td> 250 nmole ölçekte HPLC saflaştırılmış Adım B, 5.4 &#39;e bakınız </td></tr><tr><td> Bot </td><td> 5 &#39;fosfat - GAT CGG AAG AGC GGT TCA GKRY GGA ATG CCG AG 3&#39; </td><td> 250 nmole ölçekte HPLC saflaştırılmış 5 &#39;ucu Adım B, 5.4&#39; e bakınız fosforile </td></tr><tr><td> Illumina 1-454 (ileri) astar (10 uM) </td><td colspan="2"> 5 &#39;AAT GAT ACG GCG ACC ACC GAG ATC TAC ACC CTA TTK SKK GTG TGC CTT G 3&#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td> Illumina 2-454 (ters) astar (10 uM) </td><td colspan="2"> 5 &#39;CAA GKRY GAA GAC GGC ATA CGA GAT CGG TCC ATC TCA TCC CTG CGT GTC 3&#39; </td><td> 250 nmole ölçekli PCR Sınıf </td></tr><tr><td> qPCR Primer 1,1 (10 uM) </td><td colspan="2"> 5 &#39;3 saat AAT GAT ACG GCG ACC ACC GAG&#39; </td><td> 10nmole ölçekli PCR Sınıf </td></tr><tr><td> qPCR Primer 2,1 (10 uM) </td><td colspan="2"> 5 &#39;CAA GKRY GAA GAC GGC ATA CGA 3&#39; </td><td> 10nmole ölçekli PCR Sınıf </td></tr><tr><td> Illumina 3-454 dizileme astar (100 uM) </td><td colspan="2"> 5&#39;TGC GTG TTK CAT CTG TTC SKK CCC TGT CTC AG 3 &#39; </td><td> 100nmole ölçekte HPLC Saflaştırılmış </td></tr><tr><td> Illumina 4-454 dizileme astar (100 uM) </td><td colspan="2"> 5&#39;GTG SKK TGC CTA TTK SKK GTT GCG TGT CTC AG 3 &#39; </td><td> 100nmole ölçekliHPLC Saflaştırılmış </td></tr></tbody></table> Entegre DNA Technologies (IDT) ve aynı şekilde bağlayıcı ve adaptörleri hazırlamak gelen Oligolar ve adaptörler Tablo. Sipariş Oligolar önce 10 nitelendirdi. Yarı-bağlayıcı ve adaptörler -20 ° C&#39;de birkaç ay önceden hazırlanmalı ve saklanabilir

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

Genomları mikron büyüklüğünde nükleer uzaylar 7 içindeki üst düzey konformasyonları, 2, 12 benimseyerek, üç boyutlu yapıları halinde düzenlenmiştir. Böyle mimarileri rastgele değildir ve gen destekçileri ve düzenleyici unsurları 13 arasındaki etkileşimleri içerir. Belirli düzenleyici dizileri transkripsiyon faktörleri bağlayıcı transkripsiyon regülasyonu ve koordinasyon 1, 14 bir ağ beraberinde getirmektedir. Paired-end Tag Dizileme (Chia-PET) ile Kromatin Etkileşim Analizi bu yüksek mertebeli kromatin yapılarına 5,6 tanımlamak için geliştirilmiştir. Hücreler sabit ve etkileşen loci kovalent DNA-protein çapraz bağlantıları tarafından yakalanır. Non-spesifik gürültü en aza indirmek ve karmaşıklığı azaltmak, hem de kromatin etkileşim analizi özgüllüğünü artırmak için, kromatin immünopresipitasyon (çip) yakınlık ligasyon önce ilgi çekici parçaları kromatin zenginleştirmek için spesifik protein faktörlere karşı kullanılır. Yarım linkers içeren Ligasyonu sonradan bireysel kromatin kompleksleri içinde birlikte gergin DNA parçalarının çiftleri arasındaki kovalent bağlantılar oluşturur. Yarım linkers in sınırdaş MmeI restriksiyon enzimi siteleri MmeI sindirim eşleştirilmiş bitiş etiketi-linker-tag yapıları (PET) çıkarımı izin verir. Yarı-linkerler biotinlenmiş olduğu için, bu PET yapıları streptavidin-manyetik boncuklar kullanılarak arıtılır. Arıtılmış PET nesil dizileme adaptörler ve etkileşim parçaları bir katalog gibi Illumina Genom Analyzer gibi yeni nesil sequencer ile oluşturulan ile bağlandı edilir. Haritalama ve biyoinformatik analizi ardından 8 siteleri ChIP-zenginleştirilmiş bağlama ve ChIP zenginleştirilmiş kromatin etkileşimleri belirlemek için yapılır. Biz ChIP kalitesini bir Chia-PET kütüphane sonucu önemli bir rol oynar gibi özellikle Chia-PET protokolü, ChIP hazırlanması kritik yönlerini göstermek için bir video hazırladık. Protokolleri olduğundanÇok uzun, ancak kritik adımlar video gösterilir.

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

Paired-end Tag Dizileme (Chia-PET) ile Kromatin Etkileşim Analizi için kullanılan bir yöntemdir<em&gt; De novo</emTranskripsiyonel kontrolü daha iyi anlaşılması için kromatin etkileşimlerin&gt; algılama.

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

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

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Biology

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

30.5K Görüntüleme.  Agency for Science, Technology and Research, Singapore. Paired-end Tag Dizileme (Chia-PET) ile Kromatin Etkileşim Analizi için kullanılan bir yöntemdir De novo algılama.

Video: 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.

İnsan Beyninin Doku Kromatin Testi

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

Biyoloji

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.

10 nanogram Total RNA tek okuyun ve Eşleştirilmiş Sona mRNA-Seq Illumina Kütüphaneler

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.

Kullanarak Kromatin immünopresipitasyon (ChIP) Drosophila Doku

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.

RNA Saflaştırma tarafından Kromatin İzolasyon (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.

Yüksek verim Sıralama için Yüksek Kalite Kromatin Immunoprecipitation DNA Şablon üretimi (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.

Tüm montaj ovüllerindeki in Kromatin Modifikasyonu ve Nükleer Mimarlık Kantitatif, Tek hücreli Analizi için Etkin Bir Yöntem<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.

Nesil ve İnsan INO80 Kromatin Yaptırma Kompleksleri ve subcomplexes saflaştırılması

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.

Arabidopsis Protein-DNA Etkileşimleri Tespiti için Kromatin Immunoprecipitation Deneyi<em&gt; İn 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.

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 ...

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).