Ultraviolet (UV) crosslinking is a classical in vitro tool used by RNA biochemists to study RNA–protein complexes in living tissues. Scientists have successfully used CLIP (crosslinking and immunoprecipitation of RNA–protein complexes) to identify a number of target RNAs of the Nova family of neuron-specific RNA binding proteins. With the development of CLIP technology, scientists are piloting its use with several other RNA binding proteins of interest, notably the FMRP and Hu families.
Figure 1. Basic principle of CLIP. Covalent bonds are formed between proximal proteins and RNA upon exposure to ultraviolet light. These bonds only occur at the sites of direct contact and preserve RNA-protein interactions.
After ultraviolet light (UV) exposure, covalent bonds are formed between adjacent proteins and nucleic acids. Upon this special principle, CLIP has been developed as the most popular method to determine the in vivo crosslinking of RNA-protein complexes using UV. After the cross-linked cells are lysed, the target protein is isolate by immunoprecipitation (IP). In order to sequence specific primers of reverse transcription, RNA adapters are transferred to the 3' ends, radiolabeled phosphates are ligated to the 5' ends. Separate the RNA-protein complexes from free RNA using gel electrophoresis and membrane transfer. Proteinase K digestion is then performed in order to remove protein from the complexes. This step leaves a peptide at the cross-link site, allowing for the identification of the cross-linked nucleotide. After ligating RNA linkers to the 5' ends, cDNA is synthesized by RT-PCR. The last cDNA nucleotide is identified by high-throughput sequencing. Mapping the reads back to the transcriptome can identify the interaction sites.
Figure 2. Workflow of CLIP assay.
1. Ultraviolet Crosslinking of Tissue
Harvest brain, spinal cord, or other target tissues from mice. Sit the tissue in ice-cold HBSS until the harvest is complete. Set up a 500mL Stericup, remove the cellulose filter, and make a conical filter out of a sheet of 200μm nylon mesh to replace it. The resulting cell suspension is about 100mL for 10 brains. Next, transfer the tissue suspension to 50mL tubes and spin at 2500g for 5min at 4°C. Remove the supernatant and resuspend the tissue in approximate 10 times the original volume of tissue. Place the suspension in a 150mm tissue culture dish and irradiate the suspension for 400mJ/cm2. Use a dish of ice underneath the tissue suspension as you crosslink to keep the suspension cold.
Collect the irradiated suspension in a 50mL tube, then wash out each plate with an additional 5mL of HBSS, also collecting this wash. Spin down the tissue again at 2500rpm for 5min at 4°C. Resuspend the tissue in 2 times volume of the original tissue volume and pipet the solution into tubes. Spin the tubes briefly and take off the supernatant. Freeze at −80°C until use.
2. Beads Preparation
Pipet 100μL of protein A Dynabead beads solution. Place beads in magnetic stand to capture and wash beads with 500μL of 1X PXL. Repeat the wash two more times. Resuspend beads in 100μL of 1X PXL and add an appropriate amount of your anti-RNA binding protein antibody. Rock the tube for 30min to 45min at room temperature to bind the antibody to the beads. Wash the beads three times with 1X PXL.
3. Cross-linked Lysate Workup
Lyse each 100μL of cross-linked cells using 100μL of ice-cold 1X PXL. Sit the lysates on ice for 10min. Add 10μL RNasin and 10μL RQ1 DNase to each tube; incubate at 37°C for 15min. Add 1μL RNase T1 stock (1 U/μL) to the solution; incubate at 37°C for10 min, shaking at 1000rpm. Next, spin the lysates in a prechilled micro-ultracentrifuge at 90K for 25min at 4°C.
For immunoprecipitation, carefully remove the supernatant from the pelleted debris and add supernatant to one prepared tube of beads. Use about 100μL of beads per 300μL of cross-linked tissue. Rock the beads/lysate for 1h at 4°C. Wash three times with 1mL ice-cold 1X PXL and twice with 1mL ice-cold 1X PNK+.
5. Kinase the Immunoprecipitated RNA
Resuspend beads in 80μL of 1X PNK+ and add 5μL of P32 γ-ATP (γ-adenosine triphosphate) (>3000Ci/mM) and 2μL of PNK enzyme. Incubate in a thermomixer at 37°C and centrifuge 1000rpm for 20min. Finish the reaction by adding 5μL of 1mM ATP. Let the reaction continue another 5min at 37°C. Wash the beads four times with 1mL of ice-cold 1X PNK+. Resuspend the beads in 30μL of 1X PNK+ and 30μL of Novex LDS loading buffer.
6. SDS-PAGE Gel and Transfer
Load 2 wells/tube of a Novex NuPAGE 10% Bis-Tris gel. Run the gel at 150V until the dye front is at the bottom of the gel. After the gel run, transfer gel to a piece of S&S BA-85 nitrocellulose using the Novex wet transfer apparatus. After the transfer, rinse the NC filter in 1X PBS and gently blot on napkin to dry. Wrap the membrane in plastic wrap and expose to film.
7. Cut Out Crosslinked RNA–Protein Complex
In general, the RNA–protein complexes run at approximately the combined molecular weight of the protein and RNA. Using a scalpel blade, cut out this band and then cut the nitrocellulose into small pieces. Place these pieces into a single, clean tube.
8. RNA Isolation and Purification
Make a 4mg/mL proteinase K solution in 1X PK buffer; preincubate this stock at 37°C for 20min to digest any RNases. Add 200μL of this proteinase K solution to each tube of isolated NC pieces; incubate 20min at 37°C with shaking (1200rpm). Add 200μL PK/7 M urea buffer; incubate another 20min at 37°C with shaking (1200rpm).
Add 400μL RNA phenol and 130μL of CHCl3 solution. Vortex these tubes and then incubate at 37°C for 20min at 1400 rpm with shaking (1200rpm). Next, spin tubes at full speed in micro-centrifuge at 4°C or room temperature for 10min. Put the aqueous phase from each tube in a clean tube and add 50μL of 3M NaOAc, pH 5.2, and 1mL of 1:1 ethyl alcohol and isopropanol mix. Precipitate overnight at −20°C.
9. RNA Ligations
Spin down RNA at full speed in a cold centrifuge for 30min. Wash the pellet with 100μL of 75% ethyl alcohol and then dry the pellet in a SpeedVac. Count the RNA in a scintillation counter by Cerenkov counts. Use T4 RNA ligase for RNA ligations.
10. Purification of the Ligated RNA
Spin, wash, and dry RNA pellets as above. Check RNA recovery by Cerenkov counting in a scintillation counter. Pour a 20% denaturing polyacrylamide gel (1:19 bis:acrylamide, 7M urea). Resuspend the entire ligation reaction in 5μL H2O plus 5μL of formamide loading buffer. Heat the resuspended RNA at 95°C for 5min and load the entire solution on the gel; use radiolabeled PhiX markers for sizing the RNA.
After the gel run, place the gel on an old piece of film and wrap with plastic wrap. Place with a film at −80°C, generally overnight for a good signal. Using the exposed film, cut out the RNA that is greater than about 65nt. Place the gel slices in tubes; add 350μL of nucleic acid elution buffer and crush with a 1mL syringe plunger; incubate at 37°C for 30min at 1200rpm in a Thermomixer. With a cut off P1000 tip, transfer the gel slurry to a column to which you have added a 1cm glass prefilter. Spin the columns full speed in the micro-centrifuge, take the flowthrough and place it in a clean tube. Add 1mL 1:1 ethyl alcohol and isopropanol mix and 2μL of glycogen (20mg/mL stock) and precipitate overnight at −20°C.
11. cDNA and PCR
Spin, wash, and dry RNA as above; count the RNA again in a scintillation counter to quantitate yield. Resuspend the purified RNA in 9μL H2O and add 2μL of DP3 at 5pmol/μL. Heat at 65°C for 5min; chill and quick spin. The RNA is reverse transcribed into cDNA and PCR amplification was performed.
Pour a 10% denaturing polyacrylamide gel and run about 10μL of the PCR reaction on the gel; use radiolabeled markers and autoradiography gel. Cut out the major bands of about 60–100nt (and 100nt and up if you have it). Purify and precipitate the DNA as you did above for the RNA except let the DNA elute from the crushed gel slurry for at least 4h. After precipitation, resuspend the purified DNA in 5–10μL of water.
12. TOPO Cloning and Sequencing
We used three or four more rounds of PCR (one can use more if your yield is low for the first PCR). Desalt the reaction using a spin column. Then, generate the 3’ A end. Incubate at 72°C for 20min; place on ice and use immediately in the TOPO cloning reaction. Mix gently and incubate 5min at room temperature (store 3μL that you do not use in first day cloning at −20°C for potential subsequent transformation). Transform Escherichia coli as suggested by the TOPO cloning kit. Miniprep and sequence individual transformants as you would for your other sequencing reactions.
13. Database Searching
If one cannot identify the parental RNAs from which the CLIP tags are derived, the CLIP experiment is useless. The National Center for Biotechnology Information (NCBI) now has a dedicated BLAST search page for those looking for short, nearly exact matches, and it is well suited for CLIP tag identification.
Creative BioMart provides the CLIP-Seq service to analyze the RNA-protein interactions. If you have any needs, please contact our scientists to discuss details of your intend studies.
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2. Darnell R. CLIP (cross-linking and immunoprecipitation) identification of RNAs bound by a specific protein. [J]. Cold Spring Harbor Protocols, 2012, 2012(11):1146.
3. Jeon J H, Keum J H, et al. 10. Analysis of Protein-RNA Interactions with Single-Nucleotide Resolution Using iCLIP and Next-Generation Sequencing. [M]. Tag-Based Next Generation Sequencing. Wiley-VCH Verlag GmbH & Co. KGaA, 2012:153-169.