iClip - Transcriptome-breed in kaart brengen van eiwit-RNA Interacties met Individual Nucleotide resolutie

Biology
 

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Summary

De ruimtelijke ordening van RNA-bindende eiwitten op een transcript is een belangrijke determinant van post-transcriptionele regulatie. Daarom ontwikkelden we de individuele-nucleotide resolutie van UV-crosslinking en immunoprecipitatie (iClip) dat precieze genoom-brede kartering van de bindingsplaatsen van een RNA-bindend eiwit maakt.

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Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M., Ule, J. 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

De unieke samenstelling en ruimtelijke ordening van RNA-bindende eiwitten (RBPs) op een transcript gids de diverse aspecten van de post-transcriptionele regulatie 1. Daarom is een essentiële stap om het transcript regelgeving op het moleculaire niveau is het positionele informatie te verkrijgen over de bindingsplaatsen van RBPs 2.

Eiwit-RNA interacties kunnen bestudeerd worden met behulp van biochemische methoden, maar deze benaderingen niet te pakken RNA verbindend in al haar oorspronkelijke cellulaire context. De eerste pogingen om eiwit-RNA complexen studie in hun cellulaire omgeving werkzaam affiniteitszuivering of immunoprecipitatie gecombineerd met differentieel display of microarray analyse (RIP-CHIP) 3-5. Deze benaderingen waren gevoelig voor het identificeren van indirecte of niet-fysiologische interacties 6. Om de specificiteit en positionele resolutie te verhogen, een strategie om als CLIP (UV cross-linking en immunoprecipitatie) werd geïntroduceerd 7,8 verwezen. CLIP combineert UV-cross-linking van eiwitten en RNA-moleculen met strenge zuivering's waaronder denaturerende polyacrylamidegelelektroforese. In combinatie met een high-throughput sequencing technologieën, is CLIP bewezen als een krachtig instrument om eiwit-RNA-interacties op een genoom-brede schaal (aangeduid als HITS-CLIP of CLIP-seq) 9,10 te bestuderen. Onlangs werd PAR-CLIP geïntroduceerd die fotoreactief ribonucleoside analogen gebruikt voor cross-linking 11,12.

Ondanks de hoge specificiteit van de verkregen gegevens, CLIP experimenten genereren vaak cDNA-bibliotheken van de beperkte reeks complexiteit. Dit is deels te wijten aan de beperkte hoeveelheid co-gezuiverd RNA en de twee inefficiënte RNA ligatiereacties die nodig is voor bibliotheek voorbereiding. Daarnaast, primer extensie assays aangegeven dat veel cDNA's voortijdig kappen op de verknoopte nucleotide 13. Dergelijke afgekapt cDNA's zijn verloren tijdens de standaard CLIP bibliotheek voorbereiding protocol. We hebben recent ontwikkelde iClip (individuele-nucleotide resolutie CLIP), die de afgeknotte cDNA's vangt door het vervangen van een van de inefficiënte intermoleculaire RNA ligatie stappen met een meer efficiënte intramoleculaire cDNA circularization (figuur 1) 14. Belangrijk is dat sequencing de afgeknotte cDNA's geeft inzicht in de positie van de cross-link site op nucleotide resolutie. Wij hebben met succes toegepast iClip op hnRNP C deeltje organisatie op een genoom-brede schaal onderzoek en de rol ervan te beoordelen in splicing voorschrift 14.

Protocol

1. UV verknoping van weefselcultuurcellen

  1. Verwijder de media en voeg 6 ml ijskoude PBS aan cellen gekweekt in een 10 cm plaat (genoeg voor drie experimenten).
  2. Verwijder het deksel en plaats op ijs. Bestralen een keer met 150 mJ / cm 2 bij 254 nm.
  3. Oogst de cellen door schrapen met een cel lifter.
  4. Breng 2 ml celsuspensie aan elk van de drie microbuisjes. Draaien op topsnelheid voor 10 sec bij 4 ° C tot pellet cellen, verwijder supernatant.
  5. Snap-vries de cel pellets op droog ijs en bewaar bij -80 ° C tot gebruik.

2. Bead voorbereiding

  1. Voeg 100 ul van proteïne A Dynabeads (Dynal, 100,02) per experiment een nieuwe microbuisjes (Gebruik proteïne G Dynabeads voor een muis of een geit antilichamen).
  2. Was kralen 2x met lysis buffer (50 mM Tris-HCl, pH 7,4, 100 mM NaCl, 1% NP-40, 0,1% SDS; 0,5% natriumdeoxycholaat; honderdste proteaseremmer cocktail III, Calbiochem).
  3. Resuspendeer kralen in 100 ul lysisbuffer met 2-10 ug antilichaam.
  4. Draai buizen bij kamertemperatuur gedurende 30-60 minuten.
  5. Was 3x met 900 ul lysis buffer en laat in de laatste wasbeurt tot klaar om verder te gaan 4,1 stap.

3. Cellysis en gedeeltelijke RNA spijsvertering

  1. Resuspendeer de celpellet in 1 ml lysis buffer en transfer naar 1,5 ml microbuisjes.
  2. Bereid een 1 / 500 verdunning van RNase I (Ambion, AM2295). Voeg 10 pi RNase ik verwatering evenals 2 pi Turbo DNase de cel lysaat (1 / 500 RNase ik verdunningen [lage RNase] worden gebruikt voor bibliotheken voorbereiding; 1 / 50 verdunning [hoge RNase] nodig zijn om de controle voor de antilichaamspecificiteit) .
  3. Incubeer de monsters voor exact 3 min bij 37 ° C, schudden bij 1.100 tpm. Onmiddellijk over te dragen aan ijs.
  4. Spin bij 4 ° C en 22.000 g gedurende 20 minuten aan het lysaat te wissen. Zorgvuldig verzamelen supernatant (laat ongeveer 50 ui lysaat met de pellet).

4. Immunoprecipitatie

  1. Verwijder de wasbuffer van de kralen (uit stap 2.5) en voeg vervolgens de cel lysaat (vanaf stap 3.4).
  2. Draai de monsters gedurende 2 uur bij 4 ° C.
  3. Verwijder het supernatant en was de kralen 2x met 900 ul high-zout-buffer (50 mM Tris-HCl, pH 7,4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0,1% SDS; 0,5% natriumdeoxycholaat).
  4. Was 2x met 900 ul wasbuffer (20 mM Tris-HCl, pH 7,4, 10 mM MgCl2, 0,2% Tween-20).

5. Defosforylatie van RNA 3'ends

  1. Verwijder het supernatant en resuspendeer de parels in 20 pl PNK mix (15 ul water; 4 il 5x PNK pH 6,5 buffer [350mMTris-HCl, pH 6,5; 50mMMgCl 2 25mMdithiothreitol]; 0,5 ul PNK enzym; 0,5 ul RNasin [Promega]).
  2. Incubeer gedurende 20 minuten bij 37 ° C.
  3. Voeg 500 ul wasbuffer en was 1x met een hoge-zout buffer.
  4. Was 2x met wasbuffer.

6. Linker ligatie naar 3 'uiteinden RNA

  1. Verwijder voorzichtig het supernatant en resuspendeer de parels in 20 pl ligatie mix (9 ul water; 4 pi 4x ligatiebuffer [200 mMTris-HCl; 40m MM GCL 2, 40 mM dithiothreitol]; een ul RNA-ligase [NEB]; 0,5 ul RNasin [Promega]; 1,5 ul pre-adenylated linker L3 [20 uM]; 4 ul PEG400 [81170, Sigma]).
  2. Incubeer overnacht bij 16 ° C.
  3. Voeg 500 ul wasbuffer en dan 2x wassen met 1 ml high-zout buffer.
  4. Was 2x met 1 ml wasbuffer en laat in 1 ml van het tweede wassen.

7. RNA 5 'uiteinde etikettering

  1. Verwijder het supernatant en resuspendeer de kralen in 8 pl van hot PNK mix (0,4 ul PNK [NEB]; 0,8 ul 32 P-γ-ATP; 0,8 pl 10x PNK buffer [NEB]; 6 ul water).
  2. Incubeer 5 minuten bij 37 ° C.
  3. Verwijder de hete PNK mix en resuspendeer de parels in 20 pl 1x Nupage laadbuffer (Invitrogen).
  4. Incubeer op een Thermomixer bij 70 ° C gedurende 10 minuten.
  5. Direct plaats op een magneet voor het neerslaan van de lege kralen en het supernatant belasting op de gel (zie stap 8).

8. SDS-PAGE en membraan overdracht

  1. Laad de monsters op een 4-12% NuPAGE Bis-Tris Gel (Invitrogen) volgens de instructies van de fabrikant. Gebruik 0,5 l 1x MOPS lopen buffer (Invitrogen). Ook laden 5 ul van een pre-gekleurde eiwit size marker (bijvoorbeeld PAGE heerser plus, Fermentas, SM1811).
  2. Laat de gel gedurende 50 minuten op 180 V.
  3. Verwijder de gel voor-en gooi als vast afval (bevat gratis radioactief ATP).
  4. Breng de eiwit-RNA-complexen van de gel tot een nitrocellulose membraan met behulp van de Novex natte overdracht inrichting volgens de instructies van de fabrikant (Invitrogen, overdracht 1 uur bij 30 V).
  5. Na de overdracht, het membraan in PBS-buffer spoelen, dan wikkel het in Saran Wrap en blootstellen aan een Fuji film bij -80 ° C (plaats een fluorescerende sticker naast het membraan om later af te stemmen thij film en het membraan, uit te voeren blootstelling gedurende 30 minuten, 1 uur en 's nachts).

9. RNA-isolatie

  1. Isoleer de eiwit-RNA-complexen van de lage-RNase experiment met de autoradiogram uit stap 8.5 als een masker. Snijd dit stukje membraan in verschillende dunne plakjes en leg ze in een 1,5 ml microbuisjes.
  2. Voeg 200 ul PK buffer (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA) en 10 ul proteinase K (Roche, 03115828001) om het membraan stukken. Incubeer schudden bij 1100 rpm gedurende 20 min bij 37 ° C.
  3. Voeg 200 ul van PKurea buffer (100 mM Tris-HCl pH 7,4, 50 mM NaCl, 10 mM EDTA; 7 M ureum) en voor 20 min bij 37 ° C. incubeer
  4. Verzamel de oplossing en voeg deze samen met 400 ul van RNA fenol / chloroform (Ambion, 9722) om een ​​2 ml Phase Lock Gel Heavy buis (713 tot 2536, VWR).
  5. Incubeer 5 min bij 30 ° C, schudden bij 1.100 tpm. Scheid de fasen door het draaien gedurende 5 minuten bij 13.000 rpm bij kamertemperatuur.
  6. Breng de waterlaag in een nieuwe buis (pas op voor de gel contact met de pipet). Voeg 0,5 ul glycoblue (Ambion, 9510) en 40 pi 3 M natriumacetaat pH 5,5 en meng. Voeg vervolgens 1 ml 100% ethanol, meng opnieuw en neerslag gedurende de nacht bij -20 ° C.

10. Reverse transcriptie

  1. Spin gedurende 20 min bij 15000 rpm en 4 ° C. Verwijder het supernatant en was de pellet met 0,5 ml 80% ethanol.
  2. Resuspendeer de pellet in 7,25 ul RNA / primer mix (6,25 ul water; 0,5 ul Rclip primer [0.5pmol/μl]; 0,5 ul dNTP mix [10 mM]). Voor elk experiment of te repliceren, gebruik een andere Rclip primer met individuele barcode sequenties (zie 14).
  3. Incubeer 5 minuten bij 70 ° C te koelen tot 25 ° C.
  4. Voeg 2,75 ul RT mix (2 pi 5x RT buffer; 0,5 ul 0,1 M DTT, 0,25 ul Superscript III reverse transcriptase [Invitrogen]).
  5. Incubeer 5 min bij 25 ° C, 20 min bij 42 ° C, 40 min bij 50 ° C en 5 minuten bij 80 ° C te koelen tot 4 ° C.
  6. Voeg 90 ul TE-buffer, 0,5 ul glycoblue en 10 pl natriumacetaat pH 5,5 en meng. Voeg vervolgens 250 ul 100% ethanol, meng opnieuw en neerslag gedurende de nacht bij -20 ° C.

11. Gel zuivering van cDNA

  1. Spin down en was de monsters (zie 10.1), dan resuspendeer de pellets in 6 pi van water.
  2. Voeg 6 pl 2x TBE-ureum laadbuffer (Invitrogen). Warmte monsters tot 80 ° C gedurende 3 minuten direct voor het laden.
  3. Laad de monsters op een prefab 6% TBE-ureum gel (Invitrogen) en een looptijd van 40 minuten op 180 V, zoals beschreven door de fabrikant. Ook laden een laag moleculair gewicht marker voor latere snijden (zie hieronder).
  4. Snijd drie bands op 120 tot 200 nt (hoog), 85-120 nt (medium) en 70-85 nt (laag). Gebruik theupper kleurstof en de markeringen op de plastic gel steun aan excisie gids (zie figuur 3). Merk op dat de Rclip primer en de L3 volgorde zijn samen goed voor 52 nt van de CLIP-reeks.
  5. Voeg 400 ul TE en plet de gel plak in kleine stukjes met behulp van een 1 ml spuit zuiger. Incubeer schudden bij 1100 rpm gedurende 2 uur bij 37 ° C.
  6. Plaats twee 1 cm glas pre-filters (Whatman, 1.823.010) in een Costar Sphinx kolom (Corning Incorporated, 8161). Breng de vloeibare deel van het monster op de kolom. Spin voor een min bij 13000 rpm in een 1.5 ml buis.
  7. Voeg 0,5 il glycoblue en 40 pi natriumacetaat pH 5,5, en meng het monster. Voeg 1 ml 100% ethanol, meng opnieuw en neerslag gedurende de nacht bij -20 ° C.

12. Ligatie van primer aan het 5'-uiteinde van het cDNA

  1. Spin down en was de monsters (zie 10.1), dan resuspendeer de pellets in 8 pi ligatie mix (6,5 ul water; 0,8 pl 10x CircLigase Buffer II; 0,4 ul 50 mM MnCl 2, 0,3 ui, Circligase II [Epicentre]) en incubeer gedurende 1 uur bij 60 ° C.
  2. Voeg 30 ul oligo gloeien mix (26 ul water; 3 pl FastDigest Buffer [Fermentas]; 1 pi cut_oligo [10 uM]). Incubeer gedurende 1 min op 95 ° C. Vervolgens de temperatuur te verlagen om de 20 seconden met 1 ° C tot 25 ° C worden bereikt.
  3. Voeg 2 pl BamHI (Fast Fermentas) en incubeer gedurende 30 minuten bij 37 ° C.
  4. Voeg 50 ul TE en 0,5 pl glycoblue en meng. Voeg 10 ul natriumacetaat pH 5,5 en mix, voeg 250 ul 100% ethanol. Meng opnieuw en neerslag gedurende de nacht bij -20 ° C.

13. PCR-amplificatie

  1. Spin down en was de monsters (zie 10.1), dan resuspendeer de pellet in 19 ul water.
  2. Bereid de PCR-mix (19 ul cDNA, 1 ui primer mix P5/P3 Solexa, 10 pM elk; 20 ul Accuprime Supermix een enzym [Invitrogen]).
  3. Voer de volgende PCR-programma: 94 ° C gedurende 2 minuten, [94 ° C gedurende 15 sec, 65 ° C gedurende 30 sec, 68 ° C gedurende 30 sec] 25-35 cycli, 68 ° C gedurende 3 min, 4 ° C voor altijd.
  4. Mix 8 ul PCR-product met 2 pi van 5x TBE laadbuffer en de belasting op een prefab 6% TBE gel (InvitRogen). Vlekken op de gel met Sybrgreen I (Invitrogen) en te analyseren met een gel imager.
  5. De barcode op de Rclip primers laten multiplex verschillende monsters vóór het indienen van voor high throughput sequencing. Submit 15 ul van de bibliotheek voor sequencing en sla de rest.

14. Linker en primer sequenties

Pre-adenylated 3 'linker DNA:

[We bestellen het DNA-adapter van IDT en vervolgens fracties van 20μM.]

DNA

15. Representatieve resultaten:

Voorafgaand aan de opeenvolging van de iClip bibliotheek, kan het succes van het experiment te worden gecontroleerd op twee stappen: het autoradiogram van het eiwit-RNA-complex na het membraan transfer (stap 8.5) en de gel imago van de PCR-producten (stap 13.4). In de autoradiogram van de lage-RNase monsters moeten diffuse radioactiviteit te zien boven het molecuulgewicht van het eiwit (figuur 2, monster 4). Voor high-RNase samples, is deze radioactiviteit dichter bij het molecuulgewicht van het eiwit (figuur 2, voorbeeld 3) gericht. Als er geen antilichaam wordt gebruikt in de immunoprecipitatie, zou er geen signaal worden gedetecteerd (Figuur 2, monsters 1 en 2). Andere belangrijke controles voor de specificiteit van de immunoprecipitatie of weglaten UV-straling of gebruik cellen die niet uitdrukken het eiwit van belang 14.

De gel beeld van de PCR-producten (stap 13.4) moet tonen een groot bereik dat overeenkomt met het cDNA fractie (hoog, gemiddeld of laag) gezuiverd in stap 11.4 (figuur 4, lanen 4-6). Merk op dat de PCR-primers P3Solexa en P5Solexa een extra 76 nt kennis maken met de grootte van de cDNA. Als er geen antilichaam wordt gebruikt tijdens de immunoprecipitatie, mag geen overeenkomstige PCR-producten worden gedetecteerd (Figuur 4, lanen 1-3). Primer dimeer product kan verschijnen op ongeveer 140 nt.

Voor representatieve resultaten van high-throughput sequencing en de daarop volgende bioinformatica analyses zie 14.

Figuur 1
Figuur 1. Schematische weergave van de iClip protocol. Eiwit-RNA complexen covalent verknoopt in vivo met behulp van UV-bestraling (stap 1). Het eiwit van belang is samen gezuiverd met het gebonden RNA (stappen 2-5). Te zorgen voor sequentie-specifieke priming van de reverse transcriptie, is een RNA-adapter geligeerd aan het 3 'uiteinde van het RNA, terwijl het 5' einde is radioactief gelabeld (stappen 6 en 7). Cross-linked eiwit-RNA-complexen zijn gezuiverd van de gratis RNA met behulp van SDS-PAGE en membraan transfer (stap 8). Het RNA wordt teruggewonnen uit het membraan door het verteren van het eiwit met proteinase K het verlaten van een polypeptide blijven bij de cross-link nucleotide (stap 9). Reverse transcriptie (RT) afgekapt bij de resterende polypeptide en introduceert twee splitsbaar adapter regio's en barcode sequenties (stap 10). Maat selectie verwijdert gratis RT-primer voor circularization. De volgende linearisatie genereert geschikt voor de PCR-amplificatie (stap 11-15). Tot slot, high-throughput sequencing genereert leest waarin de barcode sequenties onmiddellijk gevolgd door de laatste nucleotide van het cDNA (stap 16). Aangezien dit nucleotide lokaliseert een positie stroomopwaarts van de cross-linked nucleotide, kan de bindingsplaats worden afgeleid met een hoge resolutie.

Figuur 2
Figuur 2. Autoradiogram van cross-linked hnRNP C-RNA complexen met behulp van denaturerende gelelektroforese en membraan overdracht. hnRNP C-RNA complexen werden immuno-gezuiverd van cel extracten met behulp van een antilichaam tegen hnRNP C (α hnRNP C, monsters 3 en 4). RNA werd gedeeltelijk verteerd met behulp van een laag (+) of hoge (+ +) concentratie van RNase. Complexen verschuiving naar boven van de grootte van het eiwit (40 kDa) kan worden waargenomen (voorbeeld 4). De verschuiving is minder uitgesproken bij hoge concentraties van RNase werden gebruikt (voorbeeld 3). Het radioactieve signaal verdwijnt wanneer er geen antilichaam werd gebruikt in de immunoprecipitatie (monsters 1 en 2).

Figuur 3
Figuur 3. Schematische 6% TBE-ureum gel (Invitrogen) om de excisie van iClip cDNA-producten gids. De gel wordt gerund gedurende 40 minuten op 180 V leidt tot een reproduceerbare migratiepatroon van cDNA's en kleurstoffen (licht en donker blauw) in de gel. Gebruik een scheermesje te snijden (rode lijn) de hoge (H), medium (M) en laag (L) cDNA fracties. Begin met het snijden in het midden van de licht blauwe kleurstof en vlak boven de markering op de plastic gel cassette. Verdeel het medium en de lage fracties en trim de hoge fractie ongeveer 1 cm boven de licht blauwe kleurstof. Gebruik verticaal snijdt laten leiden door de zakken en de kleurstof om de verschillende rijstroken (in dit voorbeeld 1-4) te scheiden. De marker baan (m) kan worden gekleurd en afgebeeld onder controlematen na het snijden. Fragment maten zijn aangegeven op de rechterkant.

Figuur 4
Figuur 4. Analyse van PCR-geamplificeerd iClip cDNA-bibliotheken met behulp van gelelektroforese. RNA hersteld van het membraan (figuur 1) was omgekeerd getranscribeerd en de grootte-gezuiverd met behulp van denaturerende gelelektroforese (figuur 2). Drie grootte fracties van cDNA (hoge [H]: 120 tot 200 nt, medium [M]: 85 tot 120 nt en lage [L]: 70-85 nt) teruggevonden, circularized, re-gelineariseerde en PCR-geamplificeerd. PCR-producten van verschillende grootte distributie kan worden waargenomen als gevolg van de verschillende maten van de input fracties. Omdat de PCR-primer introduceert 76 nt aan het cDNA, moeten grootte variëren tussen de 196 tot 276 nt voor hoge, 161-196 nt voor middelgrote en 146-161 nt voor de geringe omvang fracties. PCR-producten afwezig zijn wanneer er geen antilichaam werd gebruikt voor de immunoprecipitatie (lanen 1-3).

Discussion

Sinds de iClip protocol bevat een breed scala van enzymatische reacties en zuivering stappen, is het niet altijd gemakkelijk te identificeren een probleem wanneer een experiment mislukt. Met het oog op controle voor de specificiteit van geïdentificeerde RNA cross-link plaatsen, moeten een of meer negatieve controles worden gehandhaafd gedurende de gehele experiment en de daaropvolgende computationele analyses. Deze controles kunnen de no-antilichaam monster, de niet-cross-linked cellen, of immunoprecipitatie van knockout cellen of weefsel. Idealiter moeten deze controle-experimenten niet zuiveren geen eiwit-RNA-complexen, en daarom moet er geen signaal op de SDS-PAGE gel, en geen waarneembare producten na PCR-amplificatie te geven. High-throughput sequencing van deze controle bibliotheken moeten terugkeren weinig unieke sequenties. Knockdown cellen worden niet aanbevolen als een sequencing controle, omdat de resulterende sequenties nog overeen om cross-link plaatsen van hetzelfde eiwit, dat is gezuiverd van knockdown cellen in kleinere hoeveelheden.

Voorzorgsmaatregelen moeten worden genomen om besmetting met PCR-producten uit eerdere experimenten te vermijden. De beste manier om dit probleem te minimaliseren is het ruimtelijk scheiden pre-en post-PCR stappen. Idealiter zou de analyse van de PCR-producten en alle volgende stappen worden uitgevoerd in een aparte ruimte. Bovendien moet elk lid van het laboratorium gebruik maken van hun eigen set van buffers en andere reagentia. Op deze manier kunnen bronnen van besmetting gemakkelijker geïdentificeerd worden.

Disclosures

Geen belangenconflicten verklaard.

Acknowledgements

De auteurs bedanken alle leden van de Ule, Luscombe en Zupan laboratoria voor discussie en experimentele ondersteuning. Wij danken James Hadfield en Nik Matthews voor high-throughput sequencing. Wij willen er graag op wijzen dat de iClip hier beschreven methode deelt een aantal stappen met de originele CLIP protocol, ontwikkeld door Kirk Jensen en JU in het laboratorium van Robert Darnell. Dit werk werd ondersteund door de European Research Council subsidie ​​206.726-CLIP om JU en een lange-termijn Human Frontiers Science Program fellowship aan 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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