iCLIP - Bireysel Nükleotid Çözünürlük protein-RNA Etkileşimleri Transcriptome geniş Haritalama

Published 4/30/2011
71 Comments
  CITE THIS  SHARE 
Biology
 

ERRATUM NOTICE

Summary

Transkript RNA bağlayıcı proteinlerin mekansal düzenleme, post-transkripsiyonel regülasyonunda önemli bir belirleyicisidir. Bu nedenle, bir RNA bağlayıcı protein bağlama bölgeleri hassas genom haritalama sağlar bireysel nükleotid çözünürlük UV crosslinking ve immunoprecipitation (iCLIP) geliştirdi.

Cite this Article

Copy Citation

Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., et al. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J. Vis. Exp. (50), e2638, doi:10.3791/2638 (2011).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

Bir transkript RNA bağlayıcı proteinlerin benzersiz kompozisyon ve mekansal düzenleme (RBPs) post-transkripsiyonel yönetmelik 1 farklı yönlerini rehberlik eder. Bu nedenle, moleküler düzeyde transkript yönetmelik anlaşılması yönünde önemli bir adım RBPs 2 bağlanma yerleri konumsal bilgi elde etmektir .

Protein-RNA etkileşimleri biyokimyasal yöntemler kullanılarak çalışıldı, ancak bu yaklaşımların kendi doğal hücresel bağlamında bağlayıcı RNA adresi yok. İlk girişimleri kendi hücresel ortamı RNA protein kompleksleri çalışma diferansiyel görüntülemek veya mikroarray analizi (RIP-CHIP) 3-5 ile kombine yakınlık arıtma veya immunoprecipitation çalışmaktadır. Bu yaklaşımlar, dolaylı ya da fizyolojik olmayan etkileşimleri 6 belirlenmesi eğilimli . CLIP (UV çapraz bağlama ve immunoprecipitation) 7,8 tanıtıldı özgüllük ve konumsal çözünürlüğü artırmak için, bir strateji anılacaktır. CLIP UV çapraz bağlama denatüre poliakrilamid jel elektroforez de dahil olmak üzere sıkı arıtma şemaları protein ve RNA molekülleri bir araya getiriyor. Yüksek verim sıralama teknolojileri ile birlikte, CLIP, protein-RNA genom ölçekte etkileşimler (HITS-CLIP veya CLIP-seq olarak anılacaktır) 9,10 çalışmak için güçlü bir araç olarak kanıtlamıştır . Son zamanlarda, PAR-CLIP 11,12 çapraz bağlama photoreactive ribonükleozit analogları kullanan sunuldu.

Elde edilen verilerin yüksek özgüllüğü karşın, CLIP deneyler genellikle sınırlı sıra karmaşıklığı cDNA kütüphaneleri oluşturmak. Bu co-saflaştırılmış RNA kısıtlı miktarda ve kütüphane hazırlanması için gerekli olan iki verimsiz RNA ligasyonu reaksiyonlar kısmen. Buna ek olarak, astar uzatma testlerinde birçok cDNAs çapraz nükleotid 13 erken kesmek olduğunu belirtti. Bu kesilmiş cDNAs standart CLIP kütüphane hazırlık protokolü sırasında kaybolur. Biz son zamanlarda daha etkin bir molekül cDNA circularization (Şekil 1) 14 ile verimsiz intermoleküler RNA ligasyonu adımları değiştirerek kesilmiş cDNAs yakalar iCLIP (bireysel nükleotid çözünürlük CLIP) geliştirdi. Önemlisi, kesilmiş cDNAs sıralama nükleotid çözünürlükte çapraz bağ sitenin konumu içgörüler sağlar. Biz iCLIP genom ölçekte hnRNP C parçacık organizasyonun başarılı bir çalışma ve 14 ekleme düzenleme rolünü değerlendirmek için uygulanır .

Protocol

1. UV çapraz bağlayarak hücreleri, doku kültürü

  1. Ortamı çıkarın ve 10 cm plaka (üç deneyler için yeterli) yetiştirilen hücreler 6 ml buz gibi soğuk PBS ekleyin.
  2. Kapak ve buz üzerinde yer çıkarın. 150 254 nm'de mJ / cm 2 ile bir kere ışın tedavisi.
  3. Bir hücre kaldırıcı kazıma hücreleri tarafından Hasat.
  4. 2 ml hücre süspansiyonu, her üç mikrotüpler aktarın. 10 saniye boyunca en yüksek hızda Spin 4 ° C pelet hücrelerine, daha sonra süpernatant kaldırmak.
  5. Snap-, donma -80, kuru buz ve mağaza hücre pelet ° C kullanana kadar.

2. Boncuk hazırlık

  1. 100 protein ul taze bir mikrotüp Deneme başına Dynabeads (Dynal 100,02) (fare veya keçi antikorları için protein G Dynabeads kullanın).
  2. 2x boncuk lizis tamponu (100 mM NaCl,% 1 NP-40,% 0.1 SDS,% 0.5 sodyum deoksikolatın; 1 / 100 proteaz inhibitörü kokteyl III Calbiochem 50 mM Tris-HCl, pH 7.4) ile yıkayın
  3. 2-10 mg antikor ile 100 ul lizis tamponu boncuk tekrar
  4. Oda sıcaklığında 30-60 dk tüpler döndürün.
  5. 900 ul lizis tamponu ile 3x yıkayın ve son yıkama 4.1 adım ilerlemek için hazır olana kadar bekletin.

3. Hücre parçalama ve kısmi RNA sindirim

  1. 1 ml lizis tamponu ve 1.5 ml mikrotüp transfer hücre pelletini tekrar.
  2. RNaz (Ambion, AM2295) 1 / 500 dilüsyon hazırlayın. [10 ul RNaz I seyreltme yanı sıra hücre lizat 2 ul Turbo DNaz ekle (1 / 500 RNaz I dilüsyonları düşük RNaz] kütüphane hazırlanması için kullanılır; 1 / 50 dilüsyonları [yüksek RNaz] antikor özgüllüğü kontrol etmek için gerekli) .
  3. Tam 3 dakika 37 ° ° C, 1.100 rpm'de sallayarak örnekleri inkübe edin. Hemen buz transfer.
  4. 4 Spin ° C ve lizat temizlemek için 20 dakika süreyle 22.000 gr. Dikkatle (pelet ile yaklaşık 50 ul lizat bırakın) süpernatantı toplamak.

4. Immunoprecipitation

  1. Boncuk (2.5 adım) yıkama tamponu çıkarın, daha sonra hücre lizat (3.4 adım) ekleyin.
  2. 2 saat için 4 örnekleri Döndür ° C
  3. Süpernatantı atın ve 900 ul yüksek tuz tamponu (50 mM Tris-HCl, pH 7.4; 1 M NaCl, 1 mM EDTA,% 1 NP-40,% 0.1 SDS,% 0.5 sodyum deoksikolatın) 2x boncuk yıkayın.
  4. 900 ul yıkama tamponu (10 mM MgCl 2;% 0.2 Tween-20 20 mM Tris-HCl, pH 7.4) ile yıkayın 2x

5. RNA 3'ends, Defosforilasyon

  1. Süpernatant atın ve 20 ul PNK karışımı boncuklar tekrar süspansiyon (15 ul su; 4 ul 5x PNK pH 6.5 tamponu [350mMTris-HCl, pH 6.5; 50mMMgCl 2 25mMdithiothreitol]; 0.5 ul PNK enzim; 0.5 ul RNasin [Promega]).
  2. 37 20 dakika boyunca inkübe ° C.
  3. 500 ul yıkama tamponu ekleyin ve 1x yüksek tuz tamponu ile yıkayın.
  4. Yıkama tamponu ile 2x yıkayın.

6. 3 'uçları RNA Linker ligasyonu

  1. Dikkatlice süpernatant kaldırmak ve (9 ul su 20 ul ligasyon karışımı boncuklar tekrar süspansiyon; 4 ul 4x ligasyonu tampon [200 mMTris-HCl, 40 MM GCL 2; 40 mM dithiothreitol]; 1 ul RNA ligaz [NEB]; 0.5 ul RNasin [Promega]; 1.5 ul-adenylated öncesi linker L3 [20 mcM]; 4 ul PEG400 [81.170, Sigma]).
  2. 16 yaşında bir gece inkübe ° C
  3. 500 ul yıkama tamponu ekleyin ve sonra 1 ml yüksek tuz tamponu ile 2x yıkayın.
  4. Ikinci yıkama 1 ml 1 ml yıkama tamponu ile 2x ve terk yıkayın.

7. RNA 5 'sonuna etiketleme

  1. Süpernatantı ve 8 ul PNK sıcak karışımı boncuklar tekrar süspansiyon (0.4 ul PNK [NEB]; 0.8 ul 32 P-γ-ATP; 0.8 ul 10x PNK tampon [NEB]; 6 ul su).
  2. 37 5 dakika boyunca inkübe ° C.
  3. Sıcak PNK karışımı çıkarın ve 20 ul 1x Nupage yükleme tamponu (Invitrogen) boncuk tekrar süspansiyon.
  4. 70 ° C'de 10 dakika süreyle bir Thermomixer inkübe edin.
  5. Hemen boş boncuklar (bkz. adım 8) hızlandırabilir ve jel süpernatantı yüklemek için bir mıknatıs yer.

8. SDS-PAGE ve membran transferi

  1. % 4-12 NuPAGE üreticinin talimatlarına göre Bis-Tris jel (Invitrogen) numuneleri yükleyin. Tamponu (Invitrogen) 1x MOPS 0,5 l kullanın. Ayrıca bir ön-lekeli protein boyutu işaretleyici (örneğin SAYFA hükümdarı artı Fermentas, SM1811) 5 ul yük.
  2. 50 dakika için 180 jel çalıştırın V.
  3. Jel ön çıkarın ve katı atık (ücretsiz radyoaktif ATP içerir) olarak atın.
  4. Jel (Invitrogen, transferi 1 saat 30 V), üreticinin talimatlarına göre Novex ıslak transfer aparatları kullanarak bir nitroselüloz membran protein-RNA kompleksleri aktarın.
  5. Aktarımdan sonra, PBS membran durulama, Saran wrap sonra sarın ve -80 ° C (yerinde daha sonra t hizalamak için membran yanında bir floresan etiket Fuji film maruzo film ve membran) 30 dakika, 1 saat ve gece boyunca maruz kalma gerçekleştirmek.

9 - RNA izolasyonu

  1. , Bir maske olarak 8.5 adım autoradiograph kullanarak düşük RNaz deney protein-RNA kompleksleri izole edin. Bu parça membran birkaç küçük dilimler halinde kesin ve 1.5 ml mikrotüp içine yerleştirin.
  2. Ve 10 ul proteinaz K (Roche, 03115828001) 200 ul PK tamponu (10 mM EDTA; 50 mM NaCl 100 mM Tris-HCl pH: 7.4), membran parçaları ekleyin. 1.100 rpm'de 20 dakika süreyle 37 ° sallayarak inkübe ° C
  3. PKurea tamponu (50 mM NaCl; 10 mM EDTA, 100 mM Tris-HCl pH: 7.4 7 M üre) 200 ul ekleyin ve 37 ° 20 dakika boyunca inkübe ° C.
  4. Çözüm toplayın ve 2 ml Phase Lock Gel Ağır tüp (713-2536, VWR) kloroform (Ambion 9722) / fenol RNA 400 ul ile birlikte ekleyin.
  5. 1,100 rpm hızında titreme, 5 dakika 30 ° C'de inkübe edin. Oda sıcaklığında 13.000 rpm'de 5 dakika boyunca iplik aşamaları ayırın.
  6. (Jel pipet ile dokunmamaya dikkat), yeni bir tüp içine sulu aktarın. 0.5 ul glycoblue (Ambion 9510) ve 40 ul 3 M sodyum asetat, pH 5.5 ve karışımı ekleyin. Daha sonra 1 ml% 100 etanol, -20 ° C'de gece boyunca tekrar karıştırın ve çökelti

10 Ters transkripsiyon

  1. Spin 20 dakika için 15,000 rpm ve 4 ° C Süpernatantı ve pelet 0.5 ml% 80 etanol ile yıkayın.
  2. 7.25 ul RNA / astar karışımı (0.5 ul Rclip astar [0.5pmol/μl]; 0.5 ul dNTP mix [10mM] 6.25 ul su) pelletini tekrar Her deney ya da çoğaltmak için, ayrı ayrı barkod dizileri (14) içeren farklı bir Rclip astar kullanın.
  3. 5 dakika 70 ° C'de 25 ° C'ye kadar soğutma önce
  4. 2.75 ul RT karışımı (0.5 ul 0.1M DTT; 0.25 ul Üstsimge III ters transkriptaz [Invitrogen] 2 ul 5x RT tampon) ekleyin.
  5. 5 dakika 25 ° C'de 20 dakika, 42 ° C, 40 dakika 50 ° C ve 5 dakika 80 ° C 4 soğutma ° C.
  6. 90 ul TE tampon, 0.5 ul glycoblue ve 10 ul sodyum asetat, pH 5.5 ve karışımı ekleyin. Sonra 250 ul% 100 etanol, -20 ° C'de gece boyunca tekrar karıştırın ve çökelti

11. CDNA, Jel arıtma

  1. Spin ve örnekleri yıkayın (10.1), daha sonra 6 su ul pelet tekrar süspansiyon haline getirin.
  2. 6 ul 2x TBE üre yükleme tamponu (Invitrogen) ekleyin. Isı örnekleri 80 ° C doğrudan yüklemeden önce 3 dk.
  3. Bir prekast% 6 TBE üre jel (Invitrogen) numuneleri yükleyin ve üretici tarafından açıklandığı gibi 180 V 40 dakika çalıştırın. Ayrıca bir sonraki kesim için (aşağıya bakın) düşük molekül ağırlıklı işaretleyici yükleyin.
  4. 120-200 nt (yüksek), 85-120 nt (orta) ve 70-85 nt (düşük) az üç bantlarını. (Bkz. Şekil 3) eksizyon kılavuzu theupper boya ve plastik jel destek işaretleri kullanın. CLIP dizisi 52 nt Rclip astar ve birlikte L3 dizisi hesabı unutmayın.
  5. 400 ul TE ekleyin ve 1 ml şırınga pistonu kullanarak küçük parçalar halinde jel dilim ezmek. 37 az 2 saat süreyle 1.100 rpm'de sallayarak inkübe ° C
  6. Costar SpinX sütun (Corning Incorporated, 8161) içine iki adet 1 cm cam ön filtreler (whatman 1.823.010) yerleştirin. Sütun örnek sıvı kısmı aktarın. 1 dakika için 1.5 ml tüp içine 13.000 devirde dönerler.
  7. 0.5 ul glycoblue ve 40 ul sodyum asetat pH 5.5, daha sonra örnek karıştırın. 1 ml% 100 etanol, -20 ° C'de gece boyunca tekrar karıştırın ve çökelti

12. CDNA 5'end astar ligasyonu

  1. Inkübe aşağı Spin ve örnekleri yıkayın (10.1), daha sonra 8 ul ligasyon karışımı (0.8 ul 10x CircLigase Tampon II; 0.4 ul 50 mM MnCl 2; 0.3 ul Circligase II [Epicentre] 6.5 ul su) pelet tekrar süspansiyon 60 ° C'de 1 saat
  2. 30 ul oligo tavlama karıştırın (26 ul su; 3 ul FastDigest Tampon [Fermentas]; 1 ul cut_oligo [10 mcM]). 1 dakika boyunca inkübe 95 ° C Sonra her 20 saniyede 1 sıcaklığını azaltmak ° C 25 kadar ° C ulaşılır.
  3. 37 ° 2 ul BamHI (Hızlı Fermentas) ve 30 dakika boyunca inkübe ° C
  4. 50 ul TE ve 0.5 ul glycoblue ve karışımı ekleyin. 10 ul sodyum asetat, pH 5.5 ve karışımı ekleyin, daha sonra 250 ul% 100 etanol ekleyin. -20 ° C'de gece boyunca tekrar karıştırın ve çökelti

13. PCR

  1. Spin ve örnekleri yıkayın (10.1), daha sonra 19 ul su pelet tekrar süspansiyon haline getirin.
  2. (; 1 ul astar karışımı P5/P3 solexa, her 10 mcM; 20 ul Accuprime Supermix 1 enzim [Invitrogen] 19 ul cDNA) PCR karışımı hazırlayın.
  3. Aşağıdaki PCR programı çalıştırın: 25-35 döngüleri, 68 ° C 'de 3 dakika, 4 [30 saniye 15 saniye, 65 ° C - 94 ° C için 30 saniye, 68 ° C] 94 ° C' de 2 dakika süreyle, ° C sonsuza dek.
  4. 8 ul PCR ürün karması bir prekast% 6 TBE jel 5x TBE yükleme tamponu ve yük 2 ul (Invitstresin). Leke Sybrgreen I (Invitrogen) jel ve jel görüntüleme cihazı ile analiz.
  5. Rclip primerler barkod yüksek verimlilik sıralaması için göndermeden önce multipleks farklı örnekleri izin verir. Kütüphane sıralaması için 15 ul gönderin ve geri kalanı saklamak.

14. Linker ve astar dizileri

Ön adenylated 3 'linker DNA:

[20μM hacimde yapabilir sonra IDT gelen DNA bağdaştırıcısı sipariş.]

DNA

15. Temsilcisi Sonuçlar:

Deneyinin başarısı için, iCLIP kütüphane sıralanması önce iki adım izlenebilir: autoradiograph membran transferinden sonra protein-RNA kompleksi (adım 8.5) ve PCR ürünlerinin jel (adım 13.4). Düşük RNaz örnekleri autoradiograph, diffüz radyoaktivite protein (Şekil 2, örnek 4) moleküler ağırlığı üzerinde görülmelidir. Yüksek RNaz örnekleri için, bu radyoaktivite yakın proteinin moleküler ağırlığı (Şekil 2, Örnek 3) odaklanmıştır. Hiçbir antikor immunoprecipitation kullanıldığında, herhangi bir sinyal (Şekil 2, 1 ve 2 örnekleri) tespit edilmelidir. Immunoprecipitation özgüllüğü için daha önemli kontrol UV ışını veya ilgi 14 protein ifade kullanımı hücrelerin atlarsanız ya.

PCR ürünleri (adım 13.4) jel görüntü adım 11.4 (Şekil 4, şerit 4-6) saflaştırılmış cDNA fraksiyonu (yüksek, orta veya düşük) karşılık gelen bir boyut aralığı göstermelidir. PCR primerleri P3Solexa ve P5Solexa cDNA boyutu için ek bir 76 nt tanıtmak olduğunu unutmayın. Hiçbir antikor immunoprecipitation sırasında kullanılması durumunda, hiçbir karşılık gelen PCR ürünleri (Şekil 4, şerit 1-3) tespit edilmelidir. Primer-dimer ürün yaklaşık 140 nt az görünebilir.

14, yüksek verimlilik sıralama ve daha sonraki biyoinformatik analizleri temsilcisi sonuçlar için bkz .

Şekil 1
Şekil 1 iCLIP protokolü şematik. Protein-RNA kompleksleri UV ışını (adım 1) kullanarak in vivo kovalent çapraz bağlanır. Ilgi protein bağlı RNA (adım 2-5) ile birlikte arındırılır. Ters transkripsiyon sıra özel astar izin vermek için, sonunda radyoaktif (adım 6 ve 7) etiketli bir RNA adaptörü 5 iken, RNA sonu 3 bağlandı. Çapraz bağlı protein-RNA kompleksleri, SDS-PAGE ve membran transferi (adım 8) kullanarak ücretsiz RNA arınmış. RNA protein sindiren proteinaz K nükleotid çapraz bağ (adım 9) kalan bir polipeptid bırakarak membrandan kurtarıldı. Ters transkripsiyon (RT) kalan polipeptid keser ve iki cleavable adaptör bölgeler ve barkod dizileri (adım 10) tanıttı. Boyutu seçimi circularization önce ücretsiz RT astar kaldırır. Aşağıdaki doğrusallaştırma PCR amplifikasyonu için uygun şablonları (adım 11-15) üretir. Son olarak, yüksek verimli sıralama barkod dizileri hemen (adım 16) cDNA son nükleotid tarafından takip edilmektedir hangi okur üretir. Çapraz bağlı nükleotid upstream bu nükleotid bulur bir konum bu yana, bağlama site, yüksek çözünürlüğe sahip çıkarılabilir.

Şekil 2
Şekil 2 çapraz bağlantılı hnRNP C-RNA kompleksleri Autoradiograph denatüre jel elektroforezi ve membran transferi kullanarak. hnRNP C-RNA kompleksleri hücre özleri hnRNP C'ye karşı bir antikor kullanarak immüno-saflaştırılmış (α hnRNP C, örnekleri 3 ve 4). RNA kısmen düşük (+) veya yüksek kullanılarak sindirilir (+ +) konsantrasyonu RNaz. Proteinin büyüklüğü (40 kDa) yukarı doğru kayması Kompleksleri (örnek 4) görülebilir. RNaz yüksek konsantrasyonlarda (örnek 3) kullanılan zaman kayması daha az belirgindir. Herhangi bir antikor (örnekler 1 ve 2) immunoprecipitation kullanılan radyoaktif bir sinyal kaybolur.

Şekil 3
Şekil 3 şematik% 6 TBE-üre jel (Invitrogen) iCLIP cDNA ürünleri eksizyon kılavuzu. Jel, jel cDNAs ve boyalar (mavi ışık ve karanlık) tekrarlanabilir bir göç desen önde gelen 180 V 40 dakika çalıştırın. (Kırmızı çizgi), yüksek (H), orta (M), ve düşük (L) cDNA kesirler kesmek için jilet kullanın. Ortasında açık mavi boya ve plastik jel kaset işareti hemen üzerinde keserek başlayın. Orta ve düşük kesirler Böl ve yaklaşık 1 cm yukarıda açık mavi boya yüksek bir kısmını düzeltin. Farklı şerit (Bu örnekte 1-4) ayırmak için cepler ve boya tarafından yönlendirilen dikey kesikler kullanın. Işaretleyici şeritli (m) lekeli ve kontrol görüntülü olabilirkestikten sonra boyutları. Fragment boyutları sağ tarafta gösterilir.

Şekil 4
Şekil 4 jel elektroforezi kullanılarak PCR-amplifiye iCLIP cDNA kütüphaneleri analizi. RNA ters transkripsiyonu ve boyut-saflaştırılmış denatüre jel elektroforezi (Şekil 2) kullanarak membran (Şekil 1) kurtarıldı. Üç boyut fraksiyonları cDNA (yüksek [H]: 120-200 nt, orta [M]: 85-120 nt ve düşük [L]: 70-85 nt) ele geçirilmiştir circularized, yeniden lineerleştirilmiş ve PCR-amplifiye. Farklı boyut dağılımı PCR ürünleri giriş fraksiyonları farklı boyutlarda bir sonucu olarak görülebilir. PCR primer cDNA 76 nt bu yana, orta ve küçük boyut fraksiyonları için 146-161 nt boyutları, yüksek, 161-196 nt, 196-276 nt arasında aralık olmalıdır. Hiçbir antikor immunoprecipitation (şerit 1-3) için kullanılan PCR ürünleri bulunmaz.

Discussion

ICLIP protokolü enzimatik reaksiyonları ve saflaştırma adımları çok çeşitli içerdiğinden, bir deney başarısız bir sorun tespit etmek her zaman kolay değildir. Spesifite tespit RNA çapraz bağ siteleri kontrol etmek için, bir veya daha fazla negatif kontroller tam bir deney ve sonraki hesaplama analizleri boyunca muhafaza edilmelidir. Bu kontroller, no-antikor örnek, çapraz bağlantılı olmayan hücreler, ya da nakavt hücre veya doku gelen immunoprecipitation olabilir. İdeal olarak, bu kontrol deneyleri herhangi bir protein-RNA kompleksleri arındırmak gerekir, ve bu nedenle, SDS-PAGE jel üzerinde hiçbir sinyal ve PCR sonra saptanabilir ürün vermelidir. Bu kontrol kütüphaneler Yüksek throughput sıralama çok az benzersiz dizileri dönmelidir. Sonuçlanan dizileri hala, küçük miktarlarda demonte hücrelerden arındırılır aynı protein, çapraz bağ sitelerine karşılık beri demonte hücreleri, bir sıralama kontrolü olarak tavsiye edilmez.

Önlemler de önceki deneylerden elde edilen PCR ürünleri ile kirlenmesini önlemek için alınmalıdır. Bu sorunu en aza indirmek için en iyi yolu, pre-ve post-PCR adımları mekansal ayrı. İdeal olarak, PCR ürünleri ve sonraki tüm adımları analizi ayrı bir odada yapılmalıdır. Ayrıca, laboratuvarda her üye, tamponlar ve diğer reaktifler kendi kullanmalıdır. Bu şekilde, kirlenme kaynakları daha kolay tespit edilebilir.

Disclosures

Çıkar çatışması ilan etti.

Acknowledgements

Yazarlar, tartışma ve deney yardım için ULE Luscombe ve Zupan laboratuarlarında tüm üyelerine teşekkür ediyorum. James Hadfield ve Nik Matthews, yüksek verimlilik sıralaması için teşekkür ederim. Biz hisselerinin iCLIP yöntemi burada Kirk Jensen ve JU Robert Darnell laboratuar tarafından geliştirilen orijinal CLIP protokolü ile birkaç adımda açıklanan işaret etmek istiyorum. Bu çalışma, Avrupa Araştırma Konseyi hibe 206.726-JU ve JK Uzun vadeli İnsan Frontiers Lisans Programı burs CLIP tarafından desteklenen

Materials

Name Company Catalog Number Comments
For gel electrophoresis and membrane transfer we recommend t he use of XCell SureLock® Mini-Cell and XCell IIâ Blot Module Kit CE Mark (Invitrogen, EI0002), which is compatible with the use of the different precast minigels that are specified throughout the protocol. The brand and order number of all materials used is mentioned during the protocol. The list of enzymes used in the protocol is shown in the table below.
Protein A Dynabeads Invitrogen 10001D use protein G for mouse or goat antibody
RNase I Ambion AM2295 activity can change from batch to batch
T4 RNA ligase I New England Biolabs M0204S
PNK New England Biolabs M0201S
proteinase K Roche Group 03115828001
Superscript III reverse transcriptase Invitrogen 18080044
Circligase II Epicentre Biotechnologies CL9021K
FastDigest® BamHI Fermentas FD0054
AccuPrime™ SuperMix I Invitrogen 12342010 this PCR mix gives the best results in our hands

DOWNLOAD MATERIALS LIST

References

  1. Keene, J. D. RNA regulons: coordination of post-transcriptional events. Nat Rev Genet. 8, 533-543 (2007).
  2. Wang, Z., Burge, C. B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA. 14, 802-813 (2008).
  3. Trifillis, P., Day, N., Kiledjian, M. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins. RNA. 5, 1071-1082 (1999).
  4. Brooks, S. A., Rigby, W. F. Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique. Nucleic Acids Res. 28, E49-E49 (2000).
  5. Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci. 97, 14085-14090 (2000).
  6. Mili, S., Steitz, J. A. Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA. 10, 1692-1694 (2004).
  7. Ule, J. CLIP identifies Nova-regulated RNA networks in the brain. Science. 302, 1212-1215 (2003).
  8. Ule, J., Jensen, K., Mele, A., Darnell, R. B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods. 37, 376-386 (2005).
  9. Licatalosi, D. D. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 456, 464-469 (2008).
  10. Yeo, G. W. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol. 16, 130-137 (2009).
  11. Urlaub, H., Hartmuth, K., Lührmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods. 26, 170-181 (2002).
  12. König, J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol. 17, 909-915 (2010).

Erratum

Formal Correction: Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Posted by JoVE Editors on 07/14/2011. Citeable Link.

A correction was made to iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to:

"...as well as 2 μl Turbo DNase..."

instead of:

"...as well as 2 ml Turbo DNase..."

Comments

71 Comments

  1. Hi,

    First I would like to say this latest method is really neat. I also like Julian's comment at the end of the video when he said with a big smirk, "You have to perform each of the 64 steps with 100% accuracy". :D That is epic.

    On a more serious note, I am just wondering if anyone can suggest what sort of primer I should use if I want to start by cloning my insert into TOPO vector instead of doing nextGen sequencing. Any help is appreciated.

    Paul

    Reply
    Posted by: Anonymous
    June 9, 2011 - 3:47 AM
  2. Hi Paul, thanks for your fun comment! TOPO cloning dŒsn²17;t require any specific primer, so you could use the one described in the protocol. Unless you wish to do something specific, such as concatemerization of sequences before inserting them into vector. Feel free to post more questions! Jernej

    Reply
    Posted by: Anonymous
    June 11, 2011 - 4:19 PM
  3. For more iCLIP questions and answers, use the following Googledoc: http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    June 13, 2011 - 11:28 AM
  4. Hi Jernej,
    Is it possible to use a 3' linker with a phosphorylated 5' end instead of a pre-adenylated 5' end and adding some ATP during the 3' linker ligation step? Thanks. Paul

    Reply
    Posted by: Anonymous
    June 13, 2011 - 10:13 PM
  5. Yes, just follow the protocol as described in Konig et al, NSMB ²010 (PMID ²0601959). More on Googledoc.

    Reply
    Posted by: Anonymous
    June 14, 2011 - 3:48 AM
  6. Hi Jernej,

    Sorry to keep bombarding you with questions. In the supplementary section of your NSMB ²010 paper, shrimp alkaline phosphatase was used to desphosphorylate 3' ends. My understanding is that SAP can only desphosphorylate 5' ends. I am wondering if you had dephosphorylated 3' ends step using PNK before using SAP to dephosphorylate 5' ends.

    Quote from Konig et al, NSMB ²010: "For dephosphorylation of 3²4²; ends, Dynabeads were resuspended in ² µl 10&#²15; Shrimp alkaline phosphatase buffer (Promega), 17.5 µl H²O and 0.1 µl Shrimp alkaline
    phosphatase (Promega) and incubated at 37°C for 10 min with intermittent shaking (10 sec at 700 rpm followed by ²0 sec pause)."

    Thank you again for your help.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 4:04 AM
  7. We did use SAP in the NSMB protocol - it dŒsn't work as well as PNK on the 3' ends. We couldn't use PNK at the time, because PNK carryover into ligation reaction would create problems in the presence of ATP. In jove protocol, ligation reaction lacks ATP, therefore we can use PNK to dephosphorylate the 3' ends.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 6:30 PM
  8. Hi Jernej,

    On 3.² it says add ²ml Turbo DNAse into the 1.5 ml tube. I am wondering if that amount is correct.

    Reply
    Posted by: Anonymous
    July 9, 2011 - 11:45 PM
  9. Hello Paul,
    you are right, it should be two micro liters. Sorry for that, I will try to have it changed,
    Julian

    Reply
    Posted by: Anonymous
    July 10, 2011 - 6:56 AM
  10. Hi Jernej, great protocol! Just a precision, the L3 oligo is a pre-adenylated DNA or RNA oligo? Not clear as the original Clip and iClip uses RNA...

    Thanks a bunch,

    Marco

    Reply
    Posted by: Anonymous
    September 12, 2011 - 3:58 PM
  11. Hi Marco. It's a DNA oligo. Best, Jernej

    Reply
    Posted by: Anonymous
    September 12, 2011 - 4:02 PM
  12. Hi Jernej,

    I am wondering if the 3²P-ATP batch that you normally use in your lab for step 6.1 always has close to a 100% reported radioactivity. What is the lowest percentage of remaining 3²P that you can usually still get away with? I can still get some decent signal when using 3²P-ATP that has ~50-60% remaining radioactivity but my bands on the films are not as intense as the one that I see in your publications. I am trying to work out the best schedule for ordering some 3²P-ATP and starting my experiments. Thanks again.

    Paul

    Reply
    Posted by: Anonymous
    September 14, 2011 - 11:32 PM
  13. We don't use ATP if it's more than two weeks old, thus we have >50% radioactivity. But signal intensity also depends on the efficiency of crosslinking and IP,and amount of protein expression in the cells.

    Reply
    Posted by: Anonymous
    September 15, 2011 - 4:23 AM
  14. Hi Jernej,
    Thank you for the protocol. What results if I reduce the cell samples to 100-1000 (not 10*6-7 cells) ? Thanks for your reply.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 12:24 AM
  15. That would be challenging. If you have an abundant protein that cross-links well to RNA, then it might be possible. So try running the radioactive protein-RNA complex on the gel - if you good signal after overnight exposure, then it's doable.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 4:52 AM
  16. Hi, Jernej !
    Thank you for the reply. I have another questions: How stable if the RNA-RNA and RNA-Protein photocrosslinking? How to degrade these proteins or remove the photocrosslinking? Thank you a lots.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 6:36 AM
  17. Hi Jernej,

    I again have some more questions. Do you still expose the nitrocellulose membrane at -80C when using phosphoimager instead of a film? I'm also wondering what exposure time your lab uses when using a phosphorimager screen.

    Secondly, I am wondering how many libraries containing different barcodes you can run together in a single flow cells.

    Thank you again Jernej. This protocol has been extremely useful.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:49 PM
  18. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  19. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  20. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  21. Hi Jernej,

    In regards to one of the FAQs from Google docs.

    - When analysing PCR products, I see a band corresponding to the size of primer dimers, especially in the sample that was cut low from cDNA gel.

    Yes, it is common to see this band in the sample that was cut low from cDNA gel, and sometimes also in other samples. This is due to contamination from short cDNAs that only contain the sequence of RT primer. If this primer dimer is the dominant product on gel, we advise against sequencing the corresponding sample.

    I seem to be getting this short cDNA contamination all the time. Do you have any advice on how I could try to minimise the contamination? Have you ever isolated fragments of correct-size cDNA from a TBE-urea gel and sent only the isolated fragment for sequencing when you have short cDNA contaminations? Do you think that will work? I think that the concentration of L3 linker that I had used might have been too much. Thank you.

    Reply
    Posted by: Anonymous
    November 22, 2011 - 7:45 PM
  22. There are several possible reasons for this. Maybe one aspect of the protocol is not working, and therefore you are not producing any specific cDNA. If you have no cDNA input, then with enough cycles, you can amplify the primer-primer from any part of the gel. If you are using mammalian cells, try to get the protocol working first with hnRNP C or TIA with Santa cruz antibodies that we used in recent publications. Otherwise, using too much L3 can be a problem.

    Reply
    Posted by: Anonymous
    November 23, 2011 - 4:35 AM
  23. Very useful protocol. I have two questions.

    1. For dephosphorylation of RNA 3'ends, pH 6.5 PNK buffer is used, rather than the pH 7.6 buffer, provided by NEB. Have you compared these two conditions internally?
    ². In the protocol, the final PCR product is not isolated and quantitated before submitting for the sequencing. Are there any potential problems of doing these two steps? Can I isolate the PCR product and re-PCR using the same primers to get more product (for Illumina Hiseq)? Thank you.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 3:40 PM
  24. You can find more related answers in Googledoc http://goo.gl/4tSci, but short answers are also below:

    1. We haven²17;t compared conditions, but increased phophatase activity of PNK at lower pH has been reported in literature, you can read more in the Pubmed ID 1184²1²0.

    ². The PCR product needs to be quantified. We use both qPCR and bioanalyser. Normally, the products of the first PCR should look clean on the gel, otherwise it is a sign of a library that is of low complexity, and is unlikely to generate informative data. Therefore we advise against re-PCR, but it can be done as the last resource.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 4:56 PM
  25. Hi Jernej,

    I noticed you use +/- 10 multiplexed libraries; I was wondering if you knew how many are necessary for a successful run (i.e. to provide sufficient distribution for cluster identification)?

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:20 PM
  26. The way the primers are designed here, no multiplexing is necessary, because the first three nucleotides in the primer sequence are random (part of randomer = NNN).

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:28 PM
  27. I appreciate your experiment. I have some qeustions.

    In this protocol, what dŒs barcode do high-throughout squencing?

    I don't understand function of barcode



    Reply
    Posted by: seung kuk P.
    May 23, 2012 - 6:45 AM
  28. Hi,
    this might be a really naive question but I'm wondering at the UV cross linking step, when you say you irradiate once, dŒ's this mean 1 min?

    Thank you!
    Zsofi

    Reply
    Posted by: Zsofia I.
    June 18, 2012 - 1:19 PM
  29. Hello, Thank you for this helpful technique, I just have a question. My experiments protocols are: 1. UV-crosslink RNA-protein; ². Isolate the RNA-protein complex by immunopricitation; 3. Isolate the binding RNA. 3²P-labeling the binding RNA. 4. Analysis the RNA by microarray.
    Because I do not need to sequence the RNA, and I only want to isolate the binding RNA for microarray analysis after UV-crosslink RNA-protein, so I wonder whether I need to do the step 5-7 in your protocols or I could skip from step 4 to step 8 in your protocol?

    Thanks very much, I look forward to your kind reply!

    Sean

    Reply
    Posted by: xiaoyun w.
    July 22, 2012 - 9:09 PM
  30. It is unlikely you will have enough cDNA for microarray hybridisation without some kind of amplification. You can try using steps 4-8, but you could also amplify in other ways.

    Reply
    Posted by: Anonymous
    July 23, 2012 - 6:03 AM
  31. Hi Jernej,

    Is there any published article on how to analyse iCLIP's high-throughput sequencing data? I have just got my sequencing results back following steps in your protocol. I want to make sure I check with you before digging into the data. Thank you.

    Reply
    Posted by: Anonymous
    August 1, 2012 - 10:15 PM
  32. The article is not yet published, but is in preparation by Tomaz Curk ( http://www.fri.uni-lj.si/en/tomaz-curk/), who made a public server: http://icount.biolab.si/. You can contact Tomaz at tomaz.curk@fri.uni-lj.si for more information.

    Reply
    Posted by: Anonymous
    August 2, 2012 - 5:47 AM
  33. Hi,
    it is so powerful technique! But I cannot IP any protein follow protocol. Is there any difference in affinity between different antibodies and their antigen? Could you give me some advice? Maybe we could decrease concentration of SDS or sodium deoxycholate?
    Thanks, I look forward to your kind reply!
    Min

    Reply
    Posted by: Min S.
    August 5, 2012 - 11:04 PM
  34. Hi Min, you can find advice on IP googledoc http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 6, 2012 - 3:35 AM
  35. Hi Jernej,

    With the barcoding system, I am just wondering if the three random nucleotides are there for indexing purpose during Illumina sequencing run but it's not necessary for splitting the different libraries later on. For RC1, the sequencing results will be something like NNNGGTTNN.... During analysis, do you usually trim the 3-bp from the 5'-end of the results and split the different replicates after the trimming step? I have just realised this was slightly different to the barcoding system used in your NSMB paper. -paul

    Reply
    Posted by: Anonymous
    August 6, 2012 - 9:41 PM
  36. Hi Paul! You can find the answer under the topic of "Use of random barcode in data analysis" in http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 7, 2012 - 7:58 AM
  37. Hi Jernej,

    I started optimising CLIP couple of months ago and I'm at the stage that I'm convinced that I can efficiently cross link RNA to my protein (checked it by specific qRT PCR). I'm lucky because I don't need to fiddle with the IP since I've optimised before and works fine. But just to double check, after IP and western blotting a smear and a lower amount of original kDa protein is a good sing for cross linking yes?
    So my problems started at the RNase A step, I don't see any changes in size/appearance on WB after treatment... I'm convinced that my protein creates a massive complex (couple of 100 kDa) and it is because my target RNA is 10 kb to start with and there are at least 3 proteins binding to it. I'm working with a RNA virus, that's the explanation for it. I think the reason I don't see any change in kDA is because the complex dŒsn't even enter the gel to start up with. Although I used the given buffer which should break any membrane apart but the proteins are still there possibly protecting the RNA. Did you ever come across similar problems and would you have any suggestions? Also, I understand that the RNase trimming is necessary for the efficient RT step but is it a problem if the RNA is too long? What is too long? DŒs this depend on the RT enzyme used I recon or is this also important for the sequencing?

    I would greatly appreciate yur help because I'm stuck...

    Thank you,
    Zsofi

    Reply
    Posted by: Zsofia I.
    October 10, 2012 - 7:38 AM
  38. Hi Zsofi,

    For partial RNAse digestion we use RNase I (step 3). We use two different concentrations: a lower one that makes fragments with a mean between 50-100 bp and a higher concentration that fragments RNA to around 10bp. The lower one is used for preparing libraries, the higher one is used for analytical reasons.

    The RNAse step is important to (1) allow the protein RNA complex enter the Gel (²) to narrow down the crosslink site to a fragment with a size compatible with high throughput sequencing (maximum around 300 bp). So you definitely need to optimize this step for your experiments.

    If the complex you are studying is not covalently linked it should fall apart during the denaturing Gel run. Only a small fraction of your complex will have all the proteins of your complex crosslinked to the RNA at the same time since crosslinking is a very inefficient step. Therefore with the higher RNAse concentration you should be able to see a radioactive signal at the size of the protein you are studying.

    I hope that helps, best regards,
    Julian

    Reply
    Posted by: Julian K.
    October 11, 2012 - 9:49 AM
  39. Hi Julian,

    I have had some trouble with the RNase step when nuclease-ing the total lysate... In my troubleshooting efforts I read that RNase I is inhibited by 0.1% SDS, which is the concentration used in your lysis buffer. It dŒsn't seem that you guys have any problem though...do you think this is due to using an excess of RNase I or what? Just curiously confused. Thanks,

    sam

    Reply
    Posted by: Sam F.
    February 5, 2013 - 6:00 PM
  40. Hi Sam,

    in our experience the inhibition of RNase I by SDS is not an issue. You just optimize the concentration of RNase I to obtain the desired fragmentation. If you have problems doing that with your buffer conditions, you could also do the RNase digestion on the beads instead of in the lysate.

    Best,
    Julian

    Reply
    Posted by: Julian K.
    February 6, 2013 - 6:19 AM
  41. Thanks for the quick reply Julian. Your recommendation to do the "on bead" digestion is exactly what I have done and it seems to be working fine. Cheers

    Posted by: Sam F.
    February 6, 2013 - 10:12 AM
  42. Hi,

    I was wondering how many minutes have you irradiated the cells in case of HNRNP C?

    Reply
    Posted by: Niaz M.
    November 26, 2012 - 6:29 PM
  43. Hi Niaz,
    we are normally not measuring time of irradiation but the Energy per square centimeter:
    Step 1.²: ... Irradiate once with 150 mJ/cm² at ²54 nm.
    In our Stratalinker this takes 50s. However time of irradiation is not very informative here since it changes with the age or quality of the lamps, etc.
    Cheers, Julian

    Reply
    Posted by: Julian K.
    November 27, 2012 - 6:20 AM
  44. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  45. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  46. Hi Lisa,

    it is correct that the ends of P3 and P5 primers are the same. This is because of Illumina's primer design for their high throughput sequencing platform. When you look at the 3' end of the Rclip primers (after the Bamhi cleavage site) you can see that they are actually complementary to the 3'end of the L3 adapter.

    Cheers,
    Julian

    Reply
    Posted by: Julian K.
    December 18, 2012 - 10:05 AM
  47. I got it !!!
    Thank you :)

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 1:11 PM
  48. Hi
    Thanks for the protocol. I have one question that has been bothering me, though. Both the RNA ligase and PNK buffers will expose the antibody column to relatively high dithiothreitol (DTT) concentrations (10 mM and 5 mM respectively). Why dŒsn't this destroy the column by reducing the disulphide bonds holding the heavy and light antibody chains together? Have you ever tried to improve the immunoprecipitation step by attempting to minimize the DTT concentration as much as possible or is this not an issue. Any assistance would be greatly appreciated. Thanks - Greg

    Reply
    Posted by: Greg C.
    February 3, 2013 - 2:13 PM
  49. Hi Greg, we haven't seen an effect of the DTT in the buffers on the IP efficiency, it seems that the concentration is not high enough to reduce the IgG - however, it is worth testing this the first time you do IP, since it is plausible that this will vary dependent on the source of your buffers (company used for PNK and ligase), or antibodies.

    Reply
    Posted by: Anonymous
    February 6, 2013 - 2:45 AM
  50. Hi Jernej,
    Thanks for the reply. The antibody I am using is definitely sensitive to the level of DTT found in the PNK buffer and I need to limit the over-all exposure of the column to DTT as much as possible. As a result, rather than using PNK as the 3' phosphatase, I would like to use an alkaline phosphatase. I noticed that in your ²010 NSMB paper you are using Shrimp Alkaline Phosphatase and in your ²009 Methods paper you use FAST AP. Did you find that the Shrimp phosphatase is significantly better ?

    Thanks again - Greg

    Reply
    Posted by: Greg C.
    February 8, 2013 - 3:52 PM
  51. Hi Greg, we don't have any evidence to suggest that one is better than the other for the on-bead reaction. At the time we were using SAP in the lab generally since it can be heat-inactivated, so therefore we also used it for on-bead (even though here you can't heat-inactivate it on beads). So you can go ahead with either one.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:29 AM
  52. I should also add that even though we didn't compare FAST AP and SAP, we did compare SAP with PNK, and we had a lot better results with PNK. It seems that SAP is not efficient as a 3' phosphatase. So it may be better for you to determine the minimal DTT amount in the buffer that is compatible with your antibody, and then continue using it with PNK and ligase. If you use fresh DTT, 1mM is likely to be sufficient both for PNK and RNA ligase.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:39 AM
  53. Thanks, I really appreciate the advice.
    -Greg

    Posted by: Greg C.
    February 9, 2013 - 9:03 AM
  54. Hi, thanks for the awesome video. I have two questions related to the reagents:
    1. What concentration is the PEG400? (it only says 4 ul in the protocol).
    ². Under "Reverse transcription", step 6, what is the pH of the TE buffer you use? Is it pH 8?
    Thank you very much for your help. -QT

    Reply
    Posted by: Qiumin T.
    February 7, 2013 - 1:22 PM
  55. Hi QT,
    (1) we are using PEG400 from Sigma (²0²398). It is a viscous liquid.
    (²) Yes, the it is pH 8
    Cheers, Julian

    Reply
    Posted by: Julian K.
    February 7, 2013 - 4:56 PM
  56. Thank you so much Julian. I have another question. Could you recommend a protocol for doing iCLIP with mouse brain tissue? Do you know whether the tissue prep steps from this protocol ( http://ago.rockefeller.edu/Ago_HITS_CLIP_Protocol_June_²009.pdf) will work well for iCLIP as well?

    Reply
    Posted by: Anonymous
    February 13, 2013 - 5:52 PM
  57. This protocol should be fine. We also recently published a bookchapter about the iCLIP protocol which contains information on tissue samples and lots of other useful info and background:
    http://onlinelibrary.wiley.com/doi/10.100²/97835²764458².ch10/summary

    Reply
    Posted by: Julian K.
    February 14, 2013 - 5:19 AM
  58. The pre-publication version of the book chapter is available here: http://www².mrc-lmb.cam.ac.uk/groups/jule/publications/Konig_wiley.pdf.

    Reply
    Posted by: Anonymous
    February 19, 2013 - 1:54 PM
  59. I enjoyed reading about your updated iCLIP protocol in the book Tag-based Next Generation Sequencing from Wiley. I would be grateful for more details about the amount and activity of the 3²-P that you use to radioalabel the RNA. In both the book chapter and the JoVE article I see only volumes, not activities.

    In the figure for step 9, the radiolabel on the 5' end of the RNA is missing, but shouldn't it still be there? At what point in the protocol can we be reasonably sure that we are dealing with unlabeled material?

    Also, have you ever explored non-radioactive approaches to labeling, or is the sensitivity of these methods too low for the purposes of this protocol?

    Thanks!

    John

    Reply
    Posted by: John S.
    June 17, 2013 - 9:23 AM
  60. Dear John,

    with the current protocol most of the radioactivity is gone after the gel purification of the cDNA. You can increase this effect by treating the samples with RNAse after the reverse transcription (The radioactive RNA fragments then running much faster then the cDNAs in the gel). We are currently working on a protocol where we fragment the RNA by alkaline hydrolysis, which will be available soon (We want to avoid using too much RNAse at our desks).

    In addition you should always measure your samples with a Geiger counter. If your final PCRs are still hot, then you should decrease the fraction of beads that go into the labeling reaction.

    Best wishes,
    Julian

    Reply
    Posted by: Julian K.
    June 19, 2013 - 11:52 AM
  61. Hi,
    I have a question which relies on your experience with the data generated with CLIP:
    because of the UV irradiation the protein crosslinks to the RNA which even after proteinase K treatment presents an obstruction to the reverse transcriptase which therefore either skips or adds random nucleotide(s). So my question is that how long the deletions/insertions can be? Is it only one nucleotide or can also be 20?

    Thank you very much!!!

    Reply
    Posted by: Zsofia I.
    August 8, 2013 - 12:06 PM
  62. We see that >80% of cDNAs truncate at the crosslink site, and the mutations are quite rare in the remaining sequences. All we know about crosslink-induced mutations has been published here: http://www.ncbi.nlm.nih.gov/pubmed/22863408.

    Reply
    Posted by: Anonymous
    August 8, 2013 - 12:15 PM
  63. Thanks for your protocol. I have a question about the IgG background signal in the p32 labeled Western Blot. I used mouse IgG1 isotype as a control. I did not crosslink the IgG to the beads, so IgG1 stays around 50 and 25 KDa region. I do observe some radioactivity band around 50 kDa. Do you notice this in your experiments as well? Since it is close to my protein region, can you give me some suggestions to avoid this?

    Sincerely,
    Mei

    Reply
    Posted by: Xuemei Z.
    August 9, 2013 - 1:42 PM
  64. We don't get a signal in control IP. Most likely this is an RBP that non-specifically binds under your conditions. It is important to wash with high-salt buffer, and rotate the tubes for ±5min during these washes. Also, diluting the lysate before IP may help. Standard IP optimisations, basically.

    Reply
    Posted by: Anonymous
    August 9, 2013 - 2:24 PM
  65. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:29 AM
  66. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:57 AM
  67. Hi,
    I have 2 more questions. In this protocol you did not remove 5' phosphate of the RNA, can you still label the 5' side with P32 by PNK later? Another question, is it possible to just p32 label the RNA, cut the band, extract, degrade the protein and add 3' linker for RT later?

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:06 PM
  68. Normal PNK has phosphatase activity, so it can replace the 5' phosphate. The original CLIP protocol from Ule et al, Science 2003 added 3' linker after RNA extraction, but as explained in Ule et al, Methods 2005., the efficiency and purity of the protocol increases if linker is ligated on beads.

    Reply
    Posted by: Anonymous
    August 21, 2013 - 2:15 PM
  69. Thanks for this amazing protocol and your rapid and very helpful exchange here in this site.

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:26 PM
  70. Hello, thank you for this wonderful protocol.
    I have a question:
    -I get positive Radioactive signal at the right size of positive CTRL used in this protocol in the NOT UV samples, it looks exactly as I was using high RNAse condition. why?
    I am phosphorylating the protein? is it possible?
    thank you

    Reply
    Posted by: jessica c.
    February 27, 2016 - 8:45 PM
  71. Hi Jessica, you are right, if you see signal in the non-UV control, this means that the protein is getting phosphorylated in some other way. If it has a kinase domain it may even phosphorylate itself. Or maybe some kinase is getting co-purified? You could check for this by omitting PNK from the phosphorylation reaction.

    Reply
    Posted by: Jernej U.
    March 16, 2016 - 11:07 AM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Video Stats