iClip - trascrittoma livello Mappatura delle interazioni proteina-RNA con risoluzione individuale Nucleotide

Published 4/30/2011
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Summary

La disposizione spaziale di RNA-binding proteins in una trascrizione è un fattore determinante di regolazione post-trascrizionale. Pertanto, abbiamo sviluppato i singoli nucleotide reticolazione UV risoluzione e immunoprecipitazione (iClip) che permette preciso genoma mappatura dei siti di legame di una RNA-binding protein.

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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).

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Abstract

L'esclusiva composizione e la disposizione spaziale di RNA-binding proteins (RBPs) su una trascrizione guida i diversi aspetti della regolazione post-trascrizionale 1. Quindi, un passo essenziale verso la comprensione regolamento trascrizione a livello molecolare è quello di ottenere informazioni sulla posizione i siti di legame del RBPs 2.

Le interazioni proteina-RNA può essere studiato con metodi biochimici, ma questi approcci non affrontano l'RNA vincolante nel suo contesto nativo cellulare. I tentativi iniziali per lo studio delle proteine-RNA complessi nel loro ambiente cellulare impiegata purificazione di affinità o di immunoprecipitazione in combinazione con display differenziale o analisi microarray (RIP-CHIP) 3-5. Questi approcci sono stati inclini a identificare le interazioni indiretti o non fisiologica 6. Al fine di aumentare la specificità e la risoluzione di posizione, una strategia denominata CLIP (UV cross-linking e immunoprecipitazione) è stato introdotto 7,8. CLIP combina UV cross-linking delle proteine ​​e molecole di RNA con sistemi di purificazione rigorosa tra cui gel elettroforesi di poliacrilammide denaturante. In combinazione con tecnologie high-throughput sequencing, CLIP ha dimostrato come un potente strumento per studiare le interazioni proteina-RNA su un genoma scala (indicato come HITS-CLIP e CLIP-ss) 9,10. Recentemente, PAR-clip è stato introdotto che utilizza analoghi ribonucleosidi fotoreattivo per il cross-linking 11,12.

Nonostante l'alta specificità dei dati ottenuti, gli esperimenti CLIP spesso generare librerie cDNA di complessità sequenza limitata. Ciò è in parte a causa della quantità limitata di co-purificato l'RNA e le due inefficiente reazioni legatura RNA necessari per la preparazione della biblioteca. Inoltre, le analisi primer extension indicato che molti cDNA troncare prematuramente al reticolato 13 nucleotidi. Tali cDNA troncato vengono persi durante la preparazione del protocollo standard della libreria CLIP. Abbiamo recentemente sviluppato iClip (individuo-nucleotide CLIP risoluzione), che cattura il cDNA troncato sostituendo uno dei inefficiente intermolecolari passi legatura RNA con una più efficiente circularization intramolecolari cDNA (Figura 1) 14. È importante sottolineare che il sequenziamento del cDNA troncato fornisce intuizioni sulla posizione del cross-link del sito ad una risoluzione nucleotide. Abbiamo applicato con successo allo studio iClip hnRNP organizzazione delle particelle C su un genoma scala e valutare il suo ruolo nella regolazione di splicing 14.

Protocol

1. UV cross-linking di coltura dei tessuti delle cellule

  1. Rimuovere il supporto e aggiungere 6 ml ghiacciata PBS alle cellule coltivate in un piatto di 10 cm (sufficiente per tre esperimenti).
  2. Togliere il coperchio e posizionare sul ghiaccio. Irradiare una volta con 150 mJ / cm 2 a 254 nm.
  3. Raccogliere le cellule raschiando con un sollevatore di cellulare.
  4. Trasferire 2 ml di sospensione cellulare a ciascuna delle tre microtubi. Girare alla massima velocità per 10 secondi a 4 ° C per le cellule pellet, quindi rimuovere il surnatante.
  5. Snap-congelare il pellet cellulare in ghiaccio secco e conservare a -80 ° C fino al momento dell'uso.

2. Tallone preparazione

  1. Aggiungere 100 ml di proteina A Dynabeads (Dynal, 100,02) per sperimentare un microtubo fresco (Usa Dynabeads proteina G per un mouse o anticorpi di capra).
  2. Lavare perline 2x con tampone di lisi (50 mM Tris-HCl, pH 7,4, 100 mM NaCl, 1% NP-40, 0,1% SDS; deossicolato di sodio allo 0,5%, 1 / 100 della proteasi cocktail III, Calbiochem).
  3. Risospendere le sfere in 100 microlitri tampone di lisi con 2-10 mg di anticorpi.
  4. Ruotare i tubi a temperatura ambiente per 30-60 min.
  5. Lavare 3x con 900 microlitri tampone di lisi e lasciare in ultimo lavaggio fino al momento di passare al punto 4.1.

3. Lisi cellulare e parziale digestione dell'RNA

  1. Risospendere il pellet cellulare in 1 ml di tampone di lisi e il trasferimento a 1,5 microprovette ml.
  2. Preparare un 1 / 500 di diluizione di RNasi I (Ambion, AM2295). Aggiungere 10 microlitri RNasi di diluizione e 2 microlitri DNasi Turbo al lisato cellulare (1 / 500 RNase diluizioni I [bassa RNAsi] sono utilizzati per la preparazione della biblioteca; 1 / 50 diluizioni [alta RNAsi] sono indispensabili per controllare per specificità anticorpale) .
  3. Incubare i campioni per esattamente 3 minuti a 37 ° C, agitando a 1.100 giri al minuto. Trasferire immediatamente in ghiaccio.
  4. Spin a 4 ° C e 22.000 g per 20 minuti per eliminare il lisato. Raccogliere accuratamente il sopranatante (lasciare circa 50 microlitri lisato con il pellet).

4. Immunoprecipitazione

  1. Rimuovere il tampone di lavaggio dai grani (dal punto 2.5), quindi aggiungere il lisato cellulare (dal punto 3.4).
  2. Ruotare i campioni per 2 ore a 4 ° C.
  3. Eliminare il supernatante e lavare le perle di 2x con 900 microlitri di alto sale del buffer (50 mM Tris-HCl, pH 7.4, 1 M di NaCl, 1 mM EDTA, 1% NP-40, 0,1% SDS; deossicolato 0,5% di sodio).
  4. Lavare 2x con 900 microlitri di tampone di lavaggio (20 mM Tris-HCl, pH 7,4, 10 mM MgCl 2, 0.2% Tween-20).

5. Defosforilazione di 3'ends RNA

  1. Eliminare il supernatante e risospendere il mix di perle in 20 microlitri PNK (15 microlitri di acqua, 4 microlitri pH 5x PNK 6,5 buffer [350mMTris-HCl, pH 6,5; 50mMMgCl 25mMdithiothreitol 2]; enzima 0,5 microlitri PNK; 0,5 microlitri RNasin [Promega]).
  2. Incubare per 20 minuti a 37 ° C.
  3. Aggiungere 500 microlitri di buffer lavarsi e lavare 1x con alto contenuto di sale buffer.
  4. Lavare 2x con tampone di lavaggio.

6. Legatura linker per RNA 3 '

  1. Rimuovere con attenzione il sopranatante e risospendere il mix di perle in 20 microlitri legatura (9 microlitri di acqua, 4 microlitri di buffer legatura 4x [200 mMTris-HCl; MM 40m GCL 2; 40 ditiotreitolo mm]; 1 ml di RNA ligasi [NEB]; RNasin 0,5 microlitri [Promega]; 1,5 microlitri di pre-adenylated linker L3 [20 mM], 4 microlitri PEG400 [81170, Sigma]).
  2. Incubare per una notte a 16 ° C.
  3. Aggiungere 500 microlitri di buffer di lavaggio e poi lavare 2x con 1 ml di alto sale buffer.
  4. Lavare 2x con 1 ml di tampone di lavaggio e lasciare in 1 ml di secondo lavaggio.

7. Fine RNA 5 'etichettatura

  1. Rimuovere il supernatante e risospendere le sfere in 8 ml di caldo mix PNK (0,4 microlitri PNK [NEB]; 0,8 microlitri 32 P-γ-ATP; 0,8 microlitri di buffer 10x PNK [NEB]; 6 acqua microlitri).
  2. Incubare per 5 min a 37 ° C.
  3. Rimuovere il mix caldo PNK e risospendere le sfere in 20 l tampone di caricamento Nupage 1x (Invitrogen).
  4. Incubare su un Thermomixer a 70 ° C per 10 min.
  5. Immediatamente sul posto un magnete per far precipitare le perline vuoto e caricare il surnatante sul gel (vedi punto 8).

8. SDS-PAGE e trasferimento a membrana

  1. Caricare i campioni su un NuPAGE 4-12% Bis-Tris gel (Invitrogen) secondo le istruzioni del produttore. Utilizzate da 0,5 l di 1x tampone MOPS esecuzione (Invitrogen). Anche carico 5 ml di un pre-macchiato marcatore proteico dimensioni (ad esempio PAGE righello plus, Fermentas, SM1811).
  2. Esegui il gel per 50 min a 180 V.
  3. Rimuovere la parte anteriore gel ed eliminare i rifiuti solidi (contiene libero radioattivi ATP).
  4. Trasferire la proteina-RNA complessi dal gel ad una membrana di nitrocellulosa con l'apparecchiatura di trasferimento Novex umido secondo le istruzioni del produttore (Invitrogen, trasferire 1 ora a 30 V).
  5. Dopo il trasferimento, lavare la membrana in un tampone PBS, poi avvolgetelo nella pellicola trasparente e di esporla a una pellicola Fuji a -80 ° C (posto un adesivo fluorescente accanto alla membrana per allineare in seguito tche film e la membrana; eseguire esposizioni per 30 minuti, 1 ora e durante la notte).

9. RNA isolamento

  1. Isolare la proteina-RNA complessi dal basso RNasi esperimento utilizzando il autoradiograph dal punto 8.5 come una maschera. Tagliare questo pezzo di membrana in diverse fettine e metterli in un microtubo 1,5 ml.
  2. Aggiungere 200 microlitri di buffer PK (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA) e 10 microlitri proteinasi K (Roche, 03115828001) ai pezzi membrana. Incubare agitando a 1.100 rpm per 20 minuti a 37 ° C.
  3. Aggiungere 200 ml di PKurea tampone (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA, 7 M urea) e incubare per 20 minuti a 37 ° C.
  4. Raccogliere la soluzione e aggiungere insieme con 400 ml di fenolo / cloroformio (Ambion, 9722) in una fase 2 ml di tubo di blocco Gel pesanti (713-2536, VWR) RNA.
  5. Incubare per 5 minuti a 30 ° C, agitando a 1.100 giri al minuto. Separare le fasi di filatura per 5 min a 13.000 rpm a temperatura ambiente.
  6. Trasferire lo strato acquoso in un nuovo tubo (attenzione a non toccare il gel con la pipetta). Aggiungere 0,5 microlitri glycoblue (Ambion, 9510) e 40 microlitri 3 M di sodio acetato pH 5.5 e mescolare. Poi aggiungere 1 ml di etanolo al 100%, mescolare ancora e precipitare per tutta la notte a -20 ° C.

10. Trascrizione inversa

  1. Spin per 20 minuti a 15.000 rpm e 4 ° C. Rimuovere il surnatante e lavare il pellet con 0,5 ml di etanolo al 80%.
  2. Risospendere il precipitato in 7,25 microlitri di RNA / Primer Mix (6,25 microlitri di acqua, 0,5 fondo Rclip microlitri [0.5pmol/μl]; 0,5 microlitri dNTP mix [10 mM]). Per ogni esperimento o replicare, utilizzare un primer diverso Rclip contenenti sequenze di codici a barre individuale (v. 14).
  3. Incubare per 5 minuti a 70 ° C prima di raffreddamento a 25 ° C.
  4. Aggiungi 2,75 microlitri RT mix (2 microlitri di buffer 5x RT; 0,5 microlitri 0.1M DTT; 0,25 microlitri Apice III della trascrittasi inversa [Invitrogen]).
  5. Incubare 5 minuti a 25 ° C, 20 min a 42 ° C, 40 min a 50 ° C e 5 min a 80 ° C prima di raffreddamento a 4 ° C.
  6. Aggiungere 90 microlitri di buffer TE, 0,5 microlitri glycoblue e 10 microlitri acetato di sodio pH 5.5 e mescolare. Poi aggiungere 250 microlitri di etanolo al 100%, mescolare ancora e precipitare per tutta la notte a -20 ° C.

11. Gel purificazione di cDNA

  1. Spin down e lavare i campioni (vedi 10.1), poi risospendere il pellet in 6 ml di acqua.
  2. Aggiungere 6 microlitri 2x TBE-urea tampone di caricamento (Invitrogen). Campioni di calore a 80 ° C per 3 minuti prima di caricare direttamente.
  3. Caricare i campioni su un 6% prefabbricato TBE-urea gel (Invitrogen) e correre per 40 min a 180 V, come descritto dal produttore. Anche caricare un marker a basso peso molecolare per il taglio successivi (vedi sotto).
  4. Tagliare tre bande a 120-200 nt (alto), 85-120 nt (media) e 70-85 nt (basso). Usa theupper colorante ei segni sul supporto in plastica gel per guidare l'escissione (vedi figura 3). Si noti che il primer Rclip e la sequenza L3 insieme rappresentano il 52 nt della sequenza CLIP.
  5. Aggiungere 400 microlitri TE e schiacciare la fetta gel in piccoli pezzi con una siringa da 1 ml stantuffo. Incubare agitando a 1.100 giri al minuto per 2 ore a 37 ° C.
  6. Inserire due 1 centimetro di vetro pre-filtro (Whatman, 1.823.010), in una colonna Costar SpinX (Corning Incorporated, 8161). Trasferire la parte liquida del campione alla colonna. Spin per 1 minuto a 13.000 giri al minuto in una provetta da 1,5 ml.
  7. Aggiungere 0,5 microlitri glycoblue e 40 microlitri di acetato di sodio pH 5.5, poi mescolare il campione. Aggiungere 1 ml di etanolo al 100%, mescolare ancora e precipitare per tutta la notte a -20 ° C.

12. Legatura di primer per la 5'end del cDNA

  1. Spin down e lavare i campioni (vedi 10.1), poi risospendere il pellet in 8 mix legatura microlitri (6,5 microlitri di acqua; 0,8 microlitri 10x CircLigase Buffer II; 0,4 microlitri 50 mM MnCl 2; 0,3 microlitri; Circligase II [Epicentre]) e incubare per 1 ora a 60 ° C.
  2. Aggiungere 30 microlitri mix di oligo ricottura (26 microlitri di acqua, 3 Buffer FastDigest microlitri [Fermentas]; 1 ml cut_oligo [10 mM]). Incubare per 1 minuto a 95 ° C. Poi diminuire la temperatura ogni 20 sec di 1 ° C fino a 25 ° C si raggiungono.
  3. Aggiungere 2 microlitri BamHI (Fast Fermentas) e incubare per 30 minuti a 37 ° C.
  4. Aggiungere 50 microlitri TE e 0,5 microlitri glycoblue e mescolare. Aggiungere 10 microlitri di acetato di sodio pH 5.5 e mescolare, poi aggiungere 250 microlitri di etanolo al 100%. Mescolare di nuovo e precipitare per tutta la notte a -20 ° C.

13. Amplificazione PCR

  1. Spin down e lavare i campioni (vedi 10.1), poi risospendere il pellet in 19 microlitri di acqua.
  2. Preparare la miscela di PCR (19 microlitri cDNA; 1 ml fondo mix P5/P3 Solexa, 10 mM ciascuno, 20 microlitri Accuprime Supermix 1 enzima [Invitrogen]).
  3. Eseguire il seguente programma di PCR: 94 ° C per 2 minuti, [94 ° C per 15 sec, 65 ° C per 30 secondi, 68 ° C per 30 sec] 25-35 cicli, 68 ° C per 3 minuti, 4 ° C per sempre.
  4. Mix 8 microlitri prodotto di PCR con 2 ml di tampone 5x carico TBE e caricare su un 6% gel prefabbricati TBE (invitRogen). Macchiare il gel con Sybrgreen I (Invitrogen) e analizzare con un imager gel.
  5. Il codice a barre nella primer Rclip permettono ai campioni di diversi multiplex prima della presentazione per il sequenziamento throughput elevato. 15 ml di presentare la biblioteca per il sequenziamento e conservare il resto.

14. Linker e primer sequenze

DNA linker pre-adenylated 3 ':

[Ordiniamo l'adattatore DNA da IDT e poi fare aliquote di 20μM.]

DNA

15. Rappresentante dei risultati:

Prima del sequenziamento della biblioteca iClip, il successo di questo esperimento può essere monitorato in due fasi: la autoradiograph della proteina-RNA complesso dopo il trasferimento a membrana (passo 8.5) e l'immagine gel dei prodotti di PCR (passo 13,4). Nel autoradiograph del basso-RNasi campioni, la radioattività diffusa deve essere vista al di sopra del peso molecolare della proteina (Figura 2, campione 4). Per l'alta RNAsi campioni, questa radioattività è focalizzata più vicino al peso molecolare della proteina (Figura 2, campione 3). Quando non viene utilizzato anticorpi nel immunoprecipitazione, nessun segnale deve essere rilevato (Figura 2, i campioni 1 e 2). Ulteriori controlli importante per la specificità del immunoprecipitazione omettere irradiazione UV o cellule uso che non esprimono la proteina di interesse 14.

L'immagine gel dei prodotti di PCR (passo 13,4) dovrebbe mostrare un intervallo di grandezza che corrisponde alla frazione cDNA (alta, media o bassa) purificata nel passo 11,4 (Figura 4, corsie 4-6). Si noti che la PCR primer P3Solexa e P5Solexa introdurre un ulteriore 76 nt alla dimensione del cDNA. Se nessun anticorpo è utilizzato durante la immunoprecipitazione, nessun prodotto corrispondente PCR dovrebbe essere rilevato (Figura 4, corsie 1-3). Dimero prodotto primer può apparire a circa 140 nt.

Per i risultati rappresentativi di high-throughput sequencing e la successiva analisi bioinformatica vedere 14.

Figura 1
Figura 1. Rappresentazione schematica del protocollo iClip. Proteina-RNA sono complessi covalentemente reticolato in vivo, utilizzando radiazioni UV (fase 1). La proteina di interesse è purificata con l'RNA legato (passi 2-5). Per consentire la sequenza-specifica adescamento di trascrizione inversa, un adattatore RNA è legatura al 3 'del RNA, mentre il 5' è radioattivo (punti 6 e 7). Reticolato proteina-RNA complessi sono purificati da RNA gratuitamente utilizzando SDS-PAGE e trasferimento a membrana (punto 8). L'RNA è recuperato dalla membrana da digerire le proteine ​​con proteinasi K lasciando un polipeptide rimane alla cross-link nucleotide (punto 9). Trascrizione inversa (RT) tronca il polipeptide rimanente e introduce due regioni adattatore cleavable e sequenze di codici a barre (punto 10). Selezione del formato rimuove senza fondo RT prima circularization. La linearizzazione segue genera modelli adatti per l'amplificazione PCR (passi 11-15). Infine, high-throughput sequencing genera legge in cui le sequenze di codici a barre sono immediatamente seguite da nucleotide ultimo dei cDNA (passo 16). Dal momento che questo nucleotide individua una posizione a monte del reticolato nucleotide, il sito di legame si può dedurre con alta risoluzione.

Figura 2
Figura 2. Autoradiograph di cross-linked hnRNP C-RNA complessi mediante elettroforesi su gel di denaturazione e trasferimento di membrana. hnRNP C-RNA sono stati complessi immuno-purificato da estratti cellulari utilizzando un anticorpo contro hnRNP C (α hnRNP C, campioni 3 e 4). RNA è stato parzialmente digerito con bassa (+) o alto (+ +) concentrazione di RNAsi. Complessi spostando verso l'alto dalla dimensione della proteina (40 kDa) possono essere osservati (campione 4). Il cambiamento è meno pronunciato quando alte concentrazioni di RNasi sono stati utilizzati (esempio 3). Il segnale radioattivo scompare quando non anticorpale è stato utilizzato nel immunoprecipitazione (campioni 1 e 2).

Figura 3
Figura 3. Schematica 6% TBE-urea gel (Invitrogen) per guidare l'escissione di prodotti iClip cDNA. Il gel viene eseguito per 40 minuti a 180 V che porta ad un modello migratorio riproducibile di cDNA e coloranti (chiaro e blu scuro) nel gel. Utilizzare una lama di rasoio per tagliare (linea rossa) l'alto (H), medio (M) e bassa (L) frazioni cDNA. Iniziate tagliando a metà del colorante azzurro e immediatamente sopra il marchio sulla cassetta gel di plastica. Dividere le frazioni di media e bassa e tagliare la frazione alta circa 1 cm sopra il colorante azzurro. Usa tagli verticali guidati dalla tasche e il colorante per separare le corsie diverse (in questo esempio 1-4). Il vicolo marcatore (m) può essere colorate e ripreso il controllodimensioni dopo il taglio. Dimensioni frammento sono indicati sulla destra.

Figura 4
Figura 4. L'analisi di PCR-amplificato librerie iClip cDNA usando elettroforesi su gel. RNA recuperato dalla membrana (figura 1) è stata trascrizione inversa e la dimensione-purificati mediante elettroforesi su gel denaturante (Figura 2). Tre frazioni dimensione del cDNA (alta [H]: 120-200 nt, media [M]: 85-120 nt e bassa [L]: 70-85 nt) sono stati recuperati, circularized, ri-linearizzata e PCR-amplificato. I prodotti di PCR di distribuzione di diverse dimensioni si possono osservare a seguito di diverse dimensioni delle frazioni di ingresso. Dal momento che il primer PCR presenta 76 nt al cDNA, le dimensioni deve essere compresa tra 196-276 nt per alta, nt 161-196 per le medie e 146-161 nt per le frazioni bassa dimensione. I prodotti di PCR sono assenti quando non anticorpale è stato utilizzato per l'immunoprecipitazione (corsie 1-3).

Discussion

Dato che il protocollo iClip contiene una vasta gamma di reazioni enzimatiche e fasi di purificazione, non è sempre facile identificare un problema quando un esperimento fallisce. Al fine di controllo per la specificità della RNA identificato cross-link siti, uno o più controlli negativi devono essere mantenuti per tutto l'esperimento completo e la successiva analisi computazionale. Questi controlli possono essere il non-anticorpo del campione, la non cross-linked cellule, o immunoprecipitazione da cellule knockout o tessuto. Idealmente, questi esperimenti di controllo non dovrebbe purificare ogni proteina-RNA complessi, e quindi dovrebbe dare nessun segnale sul gel SDS-PAGE, e nessun prodotto rilevabile dopo amplificazione PCR. High-throughput sequencing di queste librerie di controllo dovrebbe restituire sequenze uniche pochissime. Knockdown cellule non sono raccomandati come controllo di sequenziamento, dal momento che le sequenze risultanti sempre conforme ai cross-link siti della stessa proteina, che è purificato dalle cellule atterramento in piccole quantità.

Precauzioni devono essere adottate per evitare la contaminazione con prodotti di PCR di precedenti esperimenti. Il modo migliore per minimizzare questo problema è quello di separare spazialmente fasi pre-e post-PCR. Idealmente, l'analisi dei prodotti di PCR e tutti i passi successivi devono essere eseguite in una stanza separata. Inoltre, ogni membro del laboratorio deve utilizzare il proprio set di buffer e altri reagenti. In questo modo, le fonti di contaminazione possono essere più facilmente identificati.

Disclosures

Nessun conflitto di interessi dichiarati.

Acknowledgements

Gli autori ringraziano tutti i membri dei laboratori Ule, Luscombe e Zupan di discussione e di assistenza sperimentale. Ringraziamo James Hadfield e Nik Matthews per high-throughput sequencing. Vorremmo far notare che il metodo descritto qui iClip parti diverse fasi con il protocollo clip originale, sviluppato da Kirk Jensen e JU nel laboratorio di Robert Darnell. Questo lavoro è stato sostenuto dal Consiglio europeo della ricerca concedere 206726-CLIP per JU e una a lungo termine Scienze Umane Frontiers borsa di studio del programma a JK

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

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