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iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

1, 2, 3, 3, 1, 3, 4, 2, 1

1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute

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    The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

    Date Published: 4/30/2011, Issue 50; doi: 10.3791/2638

    Cite this Article

    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).


    The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.

    Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.

    Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.


    1. UV cross-linking of tissue culture cells

    1. Remove the media and add 6 ml ice-cold PBS to cells grown in a 10 cm plate (enough for three experiments).
    2. Remove lid and place on ice. Irradiate once with 150 mJ/cm2 at 254 nm.
    3. Harvest the cells by scraping with a cell lifter.
    4. Transfer 2 ml cell suspension to each of three microtubes. Spin at top speed for 10 sec at 4°C to pellet cells, then remove supernatant.
    5. Snap-freeze the cell pellets on dry ice and store at -80°C until use.

    2. Bead preparation

    1. Add 100 μl of protein A Dynabeads (Dynal, 100.02) per experiment to a fresh microtube (Use protein G Dynabeads for a mouse or goat antibodies).
    2. Wash beads 2x with lysis buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate; 1/100 protease inhibitor cocktail III, Calbiochem).
    3. Resuspend beads in 100 μl lysis buffer with 2-10 μg antibody.
    4. Rotate tubes at room temperature for 30-60 min.
    5. Wash 3x with 900 μl lysis buffer and leave in the last wash until ready to proceed to step 4.1.

    3. Cell lysis and partial RNA digestion

    1. Resuspend the cell pellet in 1 ml lysis buffer and transfer to 1.5 ml microtubes.
    2. Prepare a 1/500 dilution of RNase I (Ambion, AM2295). Add 10 μl RNase I dilution as well as 2 μl Turbo DNase to the cell lysate (1/500 RNase I dilutions [low RNase] are used for library preparation; 1/50 dilutions [high RNase] are necessary to control for antibody specificity).
    3. Incubate the samples for exactly 3 min at 37°C, shaking at 1,100 rpm. Immediately transfer to ice.
    4. Spin at 4°C and 22,000 g for 20 min to clear the lysate. Carefully collect the supernatant (leave about 50 μl lysate with the pellet).

    4. Immunoprecipitation

    1. Remove the wash buffer from the beads (from step 2.5), then add the cell lysate (from step 3.4).
    2. Rotate the samples for 2 h at 4°C.
    3. Discard the supernatant and wash the beads 2x with 900 μl high-salt buffer (50 mM Tris-HCl, pH 7.4; 1 M NaCl; 1 mM EDTA; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate).
    4. Wash 2x with 900 μl wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20).

    5. Dephosphorylation of RNA 3'ends

    1. Discard the supernatant and resuspend the beads in 20 μl PNK mix (15 μl water; 4 μl 5x PNK pH 6.5 buffer [350mMTris-HCl, pH 6.5; 50mMMgCl2 25mMdithiothreitol]; 0.5 μl PNK enzyme; 0.5 μl RNasin [Promega]).
    2. Incubate for 20 min at 37°C.
    3. Add 500 μl wash buffer and wash 1x with high-salt buffer.
    4. Wash 2x with wash buffer.

    6. Linker ligation to RNA 3' ends

    1. Carefully remove the supernatant and resuspend the beads in 20 μl ligation mix (9 μl water; 4 μl 4x ligation buffer [200 mMTris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 μl RNA ligase [NEB]; 0.5 μl RNasin [Promega]; 1.5 μl pre-adenylated linker L3 [20 μM]; 4 μl PEG400 [81170, Sigma]).
    2. Incubate overnight at 16°C.
    3. Add 500 μl wash buffer and then wash 2x with 1 ml high-salt buffer.
    4. Wash 2x with 1 ml wash buffer and leave in 1 ml of the second wash.

    7. RNA 5' end labelling

    1. Remove the supernatant and resuspend the beads in 8 μl of hot PNK mix (0.4 μl PNK [NEB]; 0.8 μl 32P-γ-ATP; 0.8 μl 10x PNK buffer [NEB]; 6 μl water).
    2. Incubate for 5 min at 37°C.
    3. Remove the hot PNK mix and resuspend the beads in 20 μl 1x Nupage loading buffer (Invitrogen).
    4. Incubate on a thermomixer at 70°C for 10 min.
    5. Immediately place on a magnet to precipitate the empty beads and load the supernatant on the gel (see step 8).

    8. SDS-PAGE and membrane transfer

    1. Load the samples on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) according to the manufacturer's instructions. Use 0.5 l of 1x MOPS running buffer (Invitrogen). Also load 5 μl of a pre-stained protein size marker (for example PAGE ruler plus, Fermentas, SM1811).
    2. Run the gel for 50 min at 180 V.
    3. Remove the gel front and discard as solid waste (contains free radioactive ATP).
    4. Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane using the Novex wet transfer apparatus according to the manufacturer's instructions (Invitrogen, transfer 1 h at 30 V).
    5. After the transfer, rinse the membrane in PBS buffer, then wrap it in saran wrap and expose it to a Fuji film at -80°C (place a fluorescent sticker next to the membrane to later align the film and the membrane; perform exposures for 30 min, 1h and over night).

    9. RNA isolation

    1. Isolate the protein-RNA complexes from the low-RNase experiment using the autoradiograph from step 8.5 as a mask. Cut this piece of membrane into several small slices and place them into a 1.5 ml microtube.
    2. Add 200 μl PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 μl proteinase K (Roche, 03115828001) to the membrane pieces. Incubate shaking at 1,100 rpm for 20 min at 37°C.
    3. Add 200 μl of PKurea buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA; 7 M urea) and incubate for 20 min at 37°C.
    4. Collect the solution and add it together with 400 μl of RNA phenol/chloroform (Ambion, 9722) to a 2 ml Phase Lock Gel Heavy tube (713-2536, VWR).
    5. Incubate for 5 min at 30°C, shaking at 1,100 rpm. Separate the phases by spinning for 5 min at 13,000 rpm at room temperature.
    6. Transfer the aqueous layer into a new tube (be careful not to touch the gel with the pipette). Add 0.5 μl glycoblue (Ambion, 9510) and 40 μl 3 M sodium acetate pH 5.5 and mix. Then add 1 ml 100% ethanol, mix again and precipitate over night at -20°C.

    10. Reverse transcription

    1. Spin for 20 min at 15,000 rpm and 4°C. Remove the supernatant and wash the pellet with 0.5 ml 80% ethanol.
    2. Resuspend the pellet in 7.25 μl RNA/primer mix (6.25 μl water; 0.5 μl Rclip primer [0.5pmol/μl]; 0.5 μl dNTP mix [10mM]). For each experiment or replicate, use a different Rclip primer containing individual barcode sequences (see 14).
    3. Incubate for 5 min at 70°C before cooling to 25°C.
    4. Add 2.75 μl RT mix (2 μl 5x RT buffer; 0.5 μl 0.1M DTT; 0.25 μl Superscript III reverse transcriptase [Invitrogen]).
    5. Incubate 5 min at 25°C, 20 min at 42°C, 40 min at 50°C and 5 min at 80°C before cooling to 4°C.
    6. Add 90 μl TE buffer, 0.5 μl glycoblue and 10 μl sodium acetate pH 5.5 and mix. Then add 250 μl 100% ethanol, mix again and precipitate over night at -20°C.

    11. Gel purification of cDNA

    1. Spin down and wash the samples (see 10.1), then resuspend the pellets in 6 μl of water.
    2. Add 6 μl 2x TBE-urea loading buffer (Invitrogen). Heat samples to 80°C for 3 min directly before loading.
    3. Load the samples on a precast 6% TBE-urea gel (Invitrogen) and run for 40 min at 180 V as described by the manufacturer. Also load a low molecular weight marker for subsequent cutting (see below).
    4. Cut three bands at 120-200 nt (high), 85-120 nt (medium) and 70-85 nt (low). Use theupper dye and the marks on the plastic gel support to guide excision (see Figure 3). Note that the Rclip primer and the L3 sequence together account for 52 nt of the CLIP sequence.
    5. Add 400 μl TE and crush the gel slice into small pieces using a 1 ml syringe plunger. Incubate shaking at 1,100 rpm for 2 h at 37°C.
    6. Place two 1 cm glass pre-filters (Whatman, 1823010) into a Costar SpinX column (Corning Incorporated, 8161). Transfer the liquid portion of the sample to the column. Spin for 1 min at 13,000 rpm into a 1.5 ml tube.
    7. Add 0.5 μl glycoblue and 40 μl sodium acetate pH 5.5, then mix the sample. Add 1 ml 100% ethanol, mix again and precipitate over night at -20°C.

    12. Ligation of primer to the 5'end of the cDNA

    1. Spin down and wash the samples (see 10.1), then resuspend the pellets in 8 μl ligation mix (6.5 μl water; 0.8 μl 10x CircLigase Buffer II; 0.4 μl 50 mM MnCl2; 0.3 μl; Circligase II [Epicentre]) and incubate for 1 h at 60°C.
    2. Add 30 μl oligo annealing mix (26 μl water; 3 μl FastDigest Buffer [Fermentas]; 1 μl cut_oligo [10 μM]). Incubate for 1 min at 95°C. Then decrease the temperature every 20 sec by 1°C until 25°C are reached.
    3. Add 2 μl BamHI (Fast Fermentas) and incubate for 30 min at 37°C.
    4. Add 50 μl TE and 0.5 μl glycoblue and mix. Add 10 μl sodium acetate pH 5.5 and mix, then add 250 μl 100% ethanol. Mix again and precipitate over night at -20°C.

    13. PCR amplification

    1. Spin down and wash the samples (see 10.1), then resuspend the pellet in 19 μl water.
    2. Prepare the PCR mix (19 μl cDNA; 1 μl primer mix P5/P3 solexa, 10 μM each; 20 μl Accuprime Supermix 1 enzyme [Invitrogen]).
    3. Run the following PCR programme: 94°C for 2 min, [94°C for 15 sec, 65°C for 30 sec, 68°C for 30 sec]25-35 cycles, 68°C for 3 min, 4°C for ever.
    4. Mix 8 μl PCR product with 2 μl of 5x TBE loading buffer and load on a precast 6% TBE gel (Invitrogen). Stain the gel with Sybrgreen I (Invitrogen) and analyse with a gel imager.
    5. The barcode in the Rclip primers allow to multiplex different samples before submitting for high throughput sequencing. Submit 15 μl of the library for sequencing and store the rest.

    14. Linker and primer sequences

    Pre-adenylated 3' linker DNA:

    [We order the DNA adapter from IDT and then make aliquots of 20μM.]


    15. Representative Results:

    Prior to sequencing of the iCLIP library, the success of the experiment can be monitored at two steps: the autoradiograph of the protein-RNA complex after membrane transfer (step 8.5) and the gel image of the PCR products (step 13.4). In the autoradiograph of the low-RNase samples, diffuse radioactivity should be seen above the molecular weight of the protein (Figure 2, sample 4). For high-RNase samples, this radioactivity is focused closer to the molecular weight of the protein (Figure 2, sample 3). When no antibody is used in the immunoprecipitation, no signal should be detected (Figure 2, samples 1 and 2). Further important controls for specificity of the immunoprecipitation either omit UV irradiation or use cells that do not express the protein of interest14.

    The gel image of the PCR products (step 13.4) should show a size range that corresponds to the cDNA fraction (high, medium or low) purified in step 11.4 (Figure 4, lanes 4-6). Note that the PCR primers P3Solexa and P5Solexa introduce an additional 76 nt to the size of the cDNA. If no antibody is used during the immunoprecipitation, no corresponding PCR products should be detected (Figure 4, lanes 1-3). Primer dimer product can appear at about 140 nt.

    For representative results of high-throughput sequencing and subsequent bioinformatic analyses see14.

    Figure 1
    Figure 1. Schematic representation of the iCLIP protocol. Protein-RNA complexes are covalently cross-linked in vivo using UV irradiation (step 1). The protein of interest is purified together with the bound RNA (steps 2-5). To allow for sequence-specific priming of reverse transcription, an RNA adapter is ligated to the 3' end of the RNA, whereas the 5' end is radioactively labelled (steps 6 and 7). Cross-linked protein-RNA complexes are purified from free RNA using SDS-PAGE and membrane transfer (step 8). The RNA is recovered from the membrane by digesting the protein with proteinase K leaving a polypeptide remaining at the cross-link nucleotide (step 9). Reverse transcription (RT) truncates at the remaining polypeptide and introduces two cleavable adapter regions and barcode sequences (step 10). Size selection removes free RT primer before circularization. The following linearization generates suitable templates for PCR amplification (steps 11-15). Finally, high-throughput sequencing generates reads in which the barcode sequences are immediately followed by the last nucleotide of the cDNA (step 16). Since this nucleotide locates one position upstream of the cross-linked nucleotide, the binding site can be deduced with high resolution.

    Figure 2
    Figure 2. Autoradiograph of cross-linked hnRNP C-RNA complexes using denaturing gel electrophoresis and membrane transfer. hnRNP C-RNA complexes were immuno-purified from cell extracts using an antibody against hnRNP C (α hnRNP C, samples 3 and 4). RNA was partially digested using low (+) or high (++) concentration of RNase. Complexes shifting upwards from the size of the protein (40 kDa) can be observed (sample 4). The shift is less pronounced when high concentrations of RNase were used (sample 3). The radioactive signal disappears when no antibody was used in the immunoprecipitation (samples 1 and 2).

    Figure 3
    Figure 3. Schematic 6% TBE-urea gel (Invitrogen) to guide the excision of iCLIP cDNA products. The gel is run for 40 min at 180 V leading to a reproducible migration pattern of cDNAs and dyes (light and dark blue) in the gel. Use a razor blade to cut (red line) the high (H), medium (M), and low (L) cDNA fractions. Start by cutting in the middle of the light blue dye and immediately above the mark on the plastic gel cassette. Divide the medium and low fractions and trim the high fraction about 1 cm above the light blue dye. Use vertical cuts guided by the pockets and the dye to separate the different lanes (in this example 1-4). The marker lane (m) can be stained and imaged to control sizes after the cutting. Fragment sizes are indicated on the right.

    Figure 4
    Figure 4. Analysis of PCR-amplified iCLIP cDNA libraries using gel electrophoresis. RNA recovered from the membrane (Figure 1) was reverse transcribed and size-purified using denaturing gel electrophoresis (Figure 2). Three size fractions of cDNA (high [H]: 120-200 nt, medium [M]: 85-120 nt and low [L]: 70-85 nt) were recovered, circularized, re-linearized and PCR-amplified. PCR products of different size distribution can be observed as a result of the different sizes of the input fractions. Since the PCR primer introduces 76 nt to the cDNA, sizes should range between 196-276 nt for high, 161-196 nt for medium and 146-161 nt for low size fractions. PCR products are absent when no antibody was used for the immunoprecipitation (lanes 1-3).


    Since the iCLIP protocol contains a diverse range of enzymatic reactions and purification steps, it is not always easy to identify a problem when an experiment fails. In order to control for the specificity of identified RNA cross-link sites, one or more negative controls should be maintained throughout the complete experiment and subsequent computational analyses. These controls can be the no-antibody sample, the non-cross-linked cells, or immunoprecipitation from knockout cells or tissue. Ideally, these control experiments should not purify any protein-RNA complexes, and therefore should give no signal on the SDS-PAGE gel, and no detectable products after PCR amplification. High-throughput sequencing of these control libraries should return very few unique sequences. Knockdown cells are not recommended as a sequencing control, since the resulting sequences still correspond to cross-link sites of the same protein, which is purified from knockdown cells in smaller quantities.

    Precautions should also be taken to avoid contamination with PCR products from previous experiments. The best way to minimize this problem is to spatially separate pre- and post-PCR steps. Ideally, the analysis of the PCR products and all subsequent steps should be performed in a separate room. Moreover, each member of the laboratory should use their own set of buffers and other reagents. In this way, sources of contamination can be easier identified.


    No conflicts of interest declared.


    The authors thank all members of the Ule, Luscombe and Zupan laboratories for discussion and experimental assistance. We thank James Hadfield and Nik Matthews for high-throughput sequencing. We would like to point out that the iCLIP method described here shares several steps with the original CLIP protocol, developed by Kirk Jensen and J.U. in the laboratory of Robert Darnell. This work was supported by the European Research Council grant 206726-CLIP to J.U. and a Long-term Human Frontiers Science Program fellowship to J.K.


    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


    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).


    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:

    " well as 2 μl Turbo DNase..."

    instead of:

    " well as 2 ml Turbo DNase..."




    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.


    Posted by: AnonymousJune 9, 2011, 3:47 AM

    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

    Posted by: AnonymousJune 11, 2011, 4:19 PM

    For more iCLIP questions and answers, use the following Googledoc:

    Posted by: AnonymousJune 13, 2011, 11:28 AM

    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

    Posted by: AnonymousJune 13, 2011, 10:13 PM

    Yes, just follow the protocol as described in Konig et al, NSMB ²010 (PMID ²0601959). More on Googledoc.

    Posted by: AnonymousJune 14, 2011, 3:48 AM

    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.

    Posted by: AnonymousOctober 5, 2011, 4:04 AM

    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.

    Posted by: AnonymousOctober 5, 2011, 6:30 PM

    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.

    Posted by: AnonymousJuly 9, 2011, 11:45 PM

    Hello Paul,
    you are right, it should be two micro liters. Sorry for that, I will try to have it changed,

    Posted by: AnonymousJuly 10, 2011, 6:56 AM

    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,


    Posted by: AnonymousSeptember 12, 2011, 3:58 PM

    Hi Marco. It's a DNA oligo. Best, Jernej

    Posted by: AnonymousSeptember 12, 2011, 4:02 PM

    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.


    Posted by: AnonymousSeptember 14, 2011, 11:32 PM

    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.

    Posted by: AnonymousSeptember 15, 2011, 4:23 AM

    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.

    Posted by: AnonymousNovember 15, 2011, 12:24 AM

    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.

    Posted by: AnonymousNovember 15, 2011, 4:52 AM

    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.

    Posted by: AnonymousNovember 15, 2011, 6:36 AM

    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.

    Posted by: AnonymousNovember 15, 2011, 7:49 PM

    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.

    Posted by: AnonymousNovember 15, 2011, 7:53 PM

    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.

    Posted by: AnonymousNovember 15, 2011, 7:53 PM

    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.

    Posted by: AnonymousNovember 15, 2011, 7:53 PM

    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.

    Posted by: AnonymousNovember 22, 2011, 7:45 PM

    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.

    Posted by: AnonymousNovember 23, 2011, 4:35 AM

    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.

    Posted by: AnonymousJanuary 5, 2012, 3:40 PM

    You can find more related answers in Googledoc, 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.

    Posted by: AnonymousJanuary 5, 2012, 4:56 PM

    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)?

    Posted by: AnonymousMarch 29, 2012, 2:20 PM

    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).

    Posted by: AnonymousMarch 29, 2012, 2:28 PM

    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


    Posted by: seung kuk P.May 23, 2012, 6:45 AM

    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!

    Posted by: Zsofia I.June 18, 2012, 1:19 PM

    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!


    Posted by: xiaoyun w.July 22, 2012, 9:09 PM

    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.

    Posted by: AnonymousJuly 23, 2012, 6:03 AM

    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.


    Posted by: AnonymousAugust 1, 2012, 10:15 PM

    The article is not yet published, but is in preparation by Tomaz Curk (, who made a public server: You can contact Tomaz at for more information.

    Posted by: AnonymousAugust 2, 2012, 5:47 AM

    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!

    Posted by: Min S.August 5, 2012, 11:04 PM

    Hi Min, you can find advice on IP googledoc

    Posted by: AnonymousAugust 6, 2012, 3:35 AM

    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

    Posted by: AnonymousAugust 6, 2012, 9:41 PM

    Hi Paul! You can find the answer under the topic of "Use of random barcode in data analysis" in

    Posted by: AnonymousAugust 7, 2012, 7:58 AM

    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,

    Posted by: Zsofia I.October 10, 2012, 7:38 AM

    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,

    Posted by: Julian K.October 11, 2012, 9:49 AM

    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 you think this is due to using an excess of RNase I or what? Just curiously confused. Thanks,


    Posted by: Sam F.February 5, 2013, 6:00 PM

    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.


    Posted by: Julian K.February 6, 2013, 6:19 AM

    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


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

    Posted by: Niaz M.November 26, 2012, 6:29 PM

    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

    Posted by: Julian K.November 27, 2012, 6:20 AM


    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.


    Posted by: Risa K.December 18, 2012, 3:04 AM


    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.


    Posted by: Risa K.December 18, 2012, 3:04 AM

    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.


    Posted by: Julian K.December 18, 2012, 10:05 AM

    I got it !!!
    Thank you :)


    Posted by: Risa K.December 18, 2012, 1:11 PM

    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

    Posted by: Greg C.February 3, 2013, 2:13 PM

    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.

    Posted by: AnonymousFebruary 6, 2013, 2:45 AM

    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

    Posted by: Greg C.February 8, 2013, 3:52 PM

    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.

    Posted by: AnonymousFebruary 9, 2013, 5:29 AM

    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.

    Posted by: AnonymousFebruary 9, 2013, 5:39 AM

    Thanks, I really appreciate the advice.

    Posted by: Greg C.February 9, 2013, 9:03 AM

    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

    Posted by: Qiumin T.February 7, 2013, 1:22 PM

    Hi QT,
    (1) we are using PEG400 from Sigma (²0²398). It is a viscous liquid.
    (²) Yes, the it is pH 8
    Cheers, Julian

    Posted by: Julian K.February 7, 2013, 4:56 PM

    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 (²009.pdf) will work well for iCLIP as well?

    Posted by: AnonymousFebruary 13, 2013, 5:52 PM

    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:²/97835²764458².ch10/summary

    Posted by: Julian K.February 14, 2013, 5:19 AM

    The pre-publication version of the book chapter is available here: http://www²

    Posted by: AnonymousFebruary 19, 2013, 1:54 PM

    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?



    Posted by: John S.June 17, 2013, 9:23 AM

    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,

    Posted by: Julian K.June 19, 2013, 11:52 AM

    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!!!

    Posted by: Zsofia I.August 8, 2013, 12:06 PM

    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:

    Posted by: AnonymousAugust 8, 2013, 12:15 PM

    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?


    Posted by: Xuemei Z.August 9, 2013, 1:42 PM

    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.

    Posted by: AnonymousAugust 9, 2013, 2:24 PM

    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Posted by: Bhagya B.August 13, 2013, 5:29 AM

    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Posted by: Bhagya B.August 13, 2013, 5:57 AM

    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?

    Posted by: Xuemei Z.August 21, 2013, 2:06 PM

    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.

    Posted by: AnonymousAugust 21, 2013, 2:15 PM

    Thanks for this amazing protocol and your rapid and very helpful exchange here in this site.

    Posted by: Xuemei Z.August 21, 2013, 2:26 PM

    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

    Posted by: jessica c.February 27, 2016, 8:45 PM

    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.

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

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