Name: iclip - transcriptome-wide mapping of protein-rna interactions with individual nucleotide resolution
Uploaded: 2011-04-30T14:00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution at JoVE.com

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.

1. UV cross-linking of tissue culture cells
<ol>
	<li>Remove the media and add 6 ml ice-cold PBS to cells grown in a 10 cm plate (enough for three experiments).</li>
	<li>Remove lid and place on ice. Irradiate once with 150 mJ/cm2 at 254 nm.</li>
	<li>Harvest the cells by scraping with a cell lifter.</li>
	<li>Transfer 2 ml cell suspension to each of three microtubes. Spin at top speed for 10 sec at 4&deg;C to pellet cells, then remove supernatant.</li>
	<li>Snap-freeze the cell pellets on dry ice and store at -80&deg;C until use.</li>
</ol>
2. Bead preparation
<ol>
	<li>Add 100 &mu;l of protein A Dynabeads (Dynal, 100.02) per experiment to a fresh microtube (Use protein G Dynabeads for a mouse or goat antibodies).</li>
	<li>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).</li>
	<li>Resuspend beads in 100 &mu;l lysis buffer with 2-10 &mu;g antibody.</li>
	<li>Rotate tubes at room temperature for 30-60 min.</li>
	<li>Wash 3x with 900 &mu;l lysis buffer and leave in the last wash until ready to proceed to step 4.1.</li>
</ol>
3. Cell lysis and partial RNA digestion
<ol>
	<li>Resuspend the cell pellet in 1 ml lysis buffer and transfer to 1.5 ml microtubes.</li>
	<li>Prepare a 1/500 dilution of RNase I (Ambion, AM2295). Add 10 &mu;l RNase I dilution as well as 2 &#x03BC;l 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).</li>
	<li>Incubate the samples for exactly 3 min at 37&deg;C, shaking at 1,100 rpm. Immediately transfer to ice.</li>
	<li>Spin at 4&deg;C and 22,000 g for 20 min to clear the lysate. Carefully collect the supernatant (leave about 50 &mu;l lysate with the pellet).</li>
</ol>
4. Immunoprecipitation
<ol>
	<li>Remove the wash buffer from the beads (from step 2.5), then add the cell lysate (from step 3.4).</li>
	<li>Rotate the samples for 2 h at 4&deg;C.</li>
	<li>Discard the supernatant and wash the beads 2x with 900 &mu;l 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).</li>
	<li>Wash 2x with 900 &mu;l wash buffer (20 mM Tris-HCl, pH 7.4; 10 mM MgCl2; 0.2% Tween-20).</li>
</ol>
5. Dephosphorylation of RNA 3&#39;ends
<ol>
	<li>Discard the supernatant and resuspend the beads in 20 &mu;l PNK mix (15 &mu;l water; 4 &mu;l 5x PNK pH 6.5 buffer [350mMTris-HCl, pH 6.5; 50mMMgCl2 25mMdithiothreitol]; 0.5 &mu;l PNK enzyme; 0.5 &mu;l RNasin [Promega]).</li>
	<li>Incubate for 20 min at 37&deg;C.</li>
	<li>Add 500 &mu;l wash buffer and wash 1x with high-salt buffer.</li>
	<li>Wash 2x with wash buffer.</li>
</ol>
6. Linker ligation to RNA 3' ends
<ol>
	<li>Carefully remove the supernatant and resuspend the beads in 20 &mu;l ligation mix (9 &mu;l water; 4 &mu;l 4x ligation buffer [200 mMTris-HCl; 40m MM gCl2; 40 mM dithiothreitol]; 1 &mu;l RNA ligase [NEB]; 0.5 &mu;l RNasin [Promega]; 1.5 &mu;l pre-adenylated linker L3 [20 &mu;M]; 4 &mu;l PEG400 [81170, Sigma]).</li>
	<li>Incubate overnight at 16&deg;C.</li>
	<li>Add 500 &mu;l wash buffer and then wash 2x with 1 ml high-salt buffer.</li>
	<li>Wash 2x with 1 ml wash buffer and leave in 1 ml of the second wash.</li>
</ol>
7. RNA 5&#39; end labelling
<ol>
	<li>Remove the supernatant and resuspend the beads in 8 &mu;l of hot PNK mix (0.4 &mu;l PNK [NEB]; 0.8 &mu;l 32P-&gamma;-ATP; 0.8 &mu;l 10x PNK buffer [NEB]; 6 &mu;l water).</li>
	<li>Incubate for 5 min at 37&deg;C.</li>
	<li>Remove the hot PNK mix and resuspend the beads in 20 &mu;l 1x Nupage loading buffer (Invitrogen).</li>
	<li>Incubate on a thermomixer at 70&deg;C for 10 min.</li>
	<li>Immediately place on a magnet to precipitate the empty beads and load the supernatant on the gel (see step 8).</li>
</ol>
8. SDS-PAGE and membrane transfer
<ol>
	<li>Load the samples on a 4-12% NuPAGE Bis-Tris gel (Invitrogen) according to the manufacturer&#39;s instructions. Use 0.5 l of 1x MOPS running buffer (Invitrogen). Also load 5 &mu;l of a pre-stained protein size marker (for example PAGE ruler plus, Fermentas, SM1811).</li>
	<li>Run the gel for 50 min at 180 V.</li>
	<li>Remove the gel front and discard as solid waste (contains free radioactive ATP).</li>
	<li>Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane using the Novex wet transfer apparatus according to the manufacturer&#39;s instructions (Invitrogen, transfer 1 h at 30 V).</li>
	<li>After the transfer, rinse the membrane in PBS buffer, then wrap it in saran wrap and expose it to a Fuji film at -80&deg;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).</li>
</ol>
9. RNA isolation
<ol>
	<li>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.</li>
	<li>Add 200 &mu;l PK buffer (100 mM Tris-HCl pH 7.4; 50 mM NaCl; 10 mM EDTA) and 10 &mu;l proteinase K (Roche, 03115828001) to the membrane pieces. Incubate shaking at 1,100 rpm for 20 min at 37&deg;C.</li>
	<li>Add 200 &mu;l 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&deg;C.</li>
	<li>Collect the solution and add it together with 400 &mu;l of RNA phenol/chloroform (Ambion, 9722) to a 2 ml Phase Lock Gel Heavy tube (713-2536, VWR).</li>
	<li>Incubate for 5 min at 30&deg;C, shaking at 1,100 rpm. Separate the phases by spinning for 5 min at 13,000 rpm at room temperature.</li>
	<li>Transfer the aqueous layer into a new tube (be careful not to touch the gel with the pipette). Add 0.5 &mu;l glycoblue (Ambion, 9510) and 40 &mu;l 3 M sodium acetate pH 5.5 and mix. Then add 1 ml 100% ethanol, mix again and precipitate over night at -20&deg;C.</li>
</ol>
10. Reverse transcription
<ol>
	<li>Spin for 20 min at 15,000 rpm and 4&deg;C. Remove the supernatant and wash the pellet with 0.5 ml 80% ethanol.</li>
	<li>Resuspend the pellet in 7.25 &mu;l RNA/primer mix (6.25 &mu;l water; 0.5 &mu;l Rclip primer [0.5pmol/&mu;l]; 0.5 &mu;l dNTP mix [10mM]). For each experiment or replicate, use a different Rclip primer containing individual barcode sequences (see 14).</li>
	<li>Incubate for 5 min at 70&deg;C before cooling to 25&deg;C.</li>
	<li>Add 2.75 &mu;l RT mix (2 &mu;l 5x RT buffer; 0.5 &mu;l 0.1M DTT; 0.25 &mu;l Superscript III reverse transcriptase [Invitrogen]).</li>
	<li>Incubate 5 min at 25&deg;C, 20 min at 42&deg;C, 40 min at 50&deg;C and 5 min at 80&deg;C before cooling to 4&deg;C.</li>
	<li>Add 90 &mu;l TE buffer, 0.5 &mu;l glycoblue and 10 &mu;l sodium acetate pH 5.5 and mix. Then add 250 &mu;l 100% ethanol, mix again and precipitate over night at -20&deg;C.</li>
</ol>
11. Gel purification of cDNA
<ol>
	<li>Spin down and wash the samples (see 10.1), then resuspend the pellets in 6 &mu;l of water.</li>
	<li>Add 6 &mu;l 2x TBE-urea loading buffer (Invitrogen). Heat samples to 80&deg;C for 3 min directly before loading.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Add 400 &mu;l 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&deg;C.</li>
	<li>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.</li>
	<li>Add 0.5 &mu;l glycoblue and 40 &mu;l sodium acetate pH 5.5, then mix the sample. Add 1 ml 100% ethanol, mix again and precipitate over night at -20&deg;C.</li>
</ol>
12. Ligation of primer to the 5'end of the cDNA
<ol>
	<li>Spin down and wash the samples (see 10.1), then resuspend the pellets in 8 &mu;l ligation mix (6.5 &mu;l water; 0.8 &mu;l 10x CircLigase Buffer II; 0.4 &mu;l 50 mM MnCl2; 0.3 &mu;l; Circligase II [Epicentre]) and incubate for 1 h at 60&deg;C.</li>
	<li>Add 30 &mu;l oligo annealing mix (26 &mu;l water; 3 &mu;l FastDigest Buffer [Fermentas]; 1 &mu;l cut_oligo [10 &mu;M]). Incubate for 1 min at 95&deg;C. Then decrease the temperature every 20 sec by 1&deg;C until 25&deg;C are reached.</li>
	<li>Add 2 &mu;l BamHI (Fast Fermentas) and incubate for 30 min at 37&deg;C.</li>
	<li>Add 50 &mu;l TE and 0.5 &mu;l glycoblue and mix. Add 10 &mu;l sodium acetate pH 5.5 and mix, then add 250 &mu;l 100% ethanol. Mix again and precipitate over night at -20&deg;C.</li>
</ol>
13. PCR amplification
<ol>
	<li>Spin down and wash the samples (see 10.1), then resuspend the pellet in 19 &mu;l water.</li>
	<li>Prepare the PCR mix (19 &mu;l cDNA; 1 &mu;l primer mix P5/P3 solexa, 10 &mu;M each; 20 &mu;l Accuprime Supermix 1 enzyme [Invitrogen]).</li>
	<li>Run the following PCR programme: 94&deg;C for 2 min, [94&deg;C for 15 sec, 65&deg;C for 30 sec, 68&deg;C for 30 sec]25-35 cycles, 68&deg;C for 3 min, 4&deg;C for ever.</li>
	<li>Mix 8 &mu;l PCR product with 2 &mu;l 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.</li>
	<li>The barcode in the Rclip primers allow to multiplex different samples before submitting for high throughput sequencing. Submit 15 &mu;l of the library for sequencing and store the rest.</li>
</ol>
14. Linker and primer sequences
Pre-adenylated 3' linker DNA:
[We order the DNA adapter from IDT and then make aliquots of 20&mu;M.]
<img src="/files/ftp_upload/2638/2638fig0.jpg" alt="DNA" />
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.
<img src="/files/ftp_upload/2638/2638fig1.jpg" alt="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.
<img src="/files/ftp_upload/2638/2638fig2.jpg" alt="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 (&alpha; 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).
<img src="/files/ftp_upload/2638/2638fig3.jpg" alt="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.
<img src="/files/ftp_upload/2638/2638fig4.jpg" alt="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).

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	<li>Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+regulons:+coordination+of+post-transcriptional+events.">RNA regulons: coordination of post-transcriptional events.</a> Nat Rev Genet. 8, 533-543 (2007).</li><li>Wang, Z., Burge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+regulation:+from+a+parts+list+of+regulatory+elements+to+an+integrated+splicing+code.">Splicing regulation: from a parts list of regulatory elements to an integrated splicing code.</a> RNA. 14, 802-813 (2008).</li><li>Trifillis, P., Day, N., Kiledjian, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+right+RNA:+identification+of+cellular+mRNA+substrates+for+RNA-binding+proteins.">Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.</a> RNA. 5, 1071-1082 (1999).</li><li>Brooks, S. A., Rigby, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+mRNA+ligands+bound+by+the+RNA+binding+protein+hnRNP+A2+utilizing+a+novel+in+vivo+technique.">Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique.</a> Nucleic Acids Res. 28, E49-E49 (2000).</li><li>Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc Natl Acad Sci. 97, 14085-14090 (2000).</li><li>Mili, S., Steitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+reassociation+of+RNA-binding+proteins+after+cell+lysis:+implications+for+the+interpretation+of+immunoprecipitation+analyses.">Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses.</a> RNA. 10, 1692-1694 (2004).</li><li>Ule, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP+identifies+Nova-regulated+RNA+networks+in+the+brain.">CLIP identifies Nova-regulated RNA networks in the brain.</a> Science. 302, 1212-1215 (2003).</li><li>Ule, J., Jensen, K., Mele, A., Darnell, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLIP:+A+method+for+identifying+protein-RNA+interaction+sites+in+living+cells.">CLIP: A method for identifying protein-RNA interaction sites in living cells.</a> Methods. 37, 376-386 (2005).</li><li>Licatalosi, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HITS-CLIP+yields+genome-wide+insights+into+brain+alternative+RNA+processing.">HITS-CLIP yields genome-wide insights into brain alternative RNA processing.</a> Nature. 456, 464-469 (2008).</li><li>Yeo, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+code+for+the+FOX2+splicing+regulator+revealed+by+mapping+RNA-protein+interactions+in+stem+cells.">An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells.</a> Nat Struct Mol Biol. 16, 130-137 (2009).</li><li>Urlaub, H., Hartmuth, K., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-tracked+approach+to+analyze+RNA-protein+crosslinking+sites+in+native,+nonlabeled+small+nuclear+ribonucleoprotein+particles.">A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles.</a> Methods. 26, 170-181 (2002).</li><li>K&#246;nig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iCLIP+reveals+the+function+of+hnRNP+particles+in+splicing+at+individual+nucleotide+resolution.">iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.</a> Nat Struct Mol Biol. 17, 909-915 (2010).</li></ol>

No conflicts of interest declared.

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.

A correction was made to <a href="http://www.jove.com/details.php?id=2638">iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution</a>. 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 &#x03BC;l Turbo DNase..." instead of: "...as well as 2 ml Turbo DNase..."

A correction to the article iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution was made.

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

For gel electrophoresis and membrane transfer we recommend t he use of XCell SureLock&reg; Mini-Cell and XCell II&trade; 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. 
<table border="1">
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue #</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Protein A Dynabeads</td>
 <td>Invitrogen</td>
 <td>10001D</td>
 <td>use protein G for mouse or goat antibody</td>
 </tr>
 <tr>
 <td>RNase I </td>
 <td>Ambion</td>
 <td>AM2295</td>
 <td>activity can change from batch to batch</td>
 </tr>
 <tr>
 <td>T4 RNA ligase I</td>
 <td>NEB</td>
 <td>M0204S</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>PNK</td>
 <td>NEB</td>
 <td>M0201S</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>proteinase K</td>
 <td>Roche</td>
 <td>03115828001</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>Superscript III reverse transcriptase </td>
 <td>Invitrogen</td>
 <td>18080044</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>Circligase II </td>
 <td>Epicentre</td>
 <td>CL9021K</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>FastDigest&reg; BamHI</td>
 <td>Fermentas</td>
 <td>FD0054</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>AccuPrime&trade; SuperMix I</td>
 <td>Invitrogen</td>
 <td>12342010</td>
 <td>this PCR mix gives the best results in our hands</td>
 </tr>
</table>

iclip - transcriptome-wide mapping of protein-rna interactions with individual nucleotide resolution

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.

Watch this Scientific Journal Video about iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution at JoVE.com

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

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.

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

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Research

JoVE Journal

Biology

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Hui Zhang and Niko G. Gubernator contributed equally to this work.

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

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

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

High-resolution Optical Mapping of the Mouse Sino-atrial Node

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.