The overall goal of the following experiment is to obtain a transcriptome wide map of the binding sites of an RNA binding protein at individual nucleotide resolution. This is achieved by UV irradiation of living cells to covalently cross-link proteins and RNA in vivo as a second step. The RNA binding protein is immuno purified under stringent conditions in order to recover the cross-linked RNA fragments.
Next reverse transcription generates CDNAs that often truncate at the attached peptide, which preserves the information on the cross-link position within the RNA Results are obtained that show all binding sites within the transcriptome based on high throughput sequencing of the cDNA library. This method can help answer key questions in RNA biology such as how the interplay of different RNA onic proteins regulates RNA processing. The main advantage of this technique is that we increase both the performance and the resolution of our transcriptome wide maps of protein RNA interactions To be to begin the procedure of UV cross-linking tissue culture cells.
Remove the medium from a 10 centimeter plate of healer cells. Add six milliliters of ice cold PBS and place the plates on ice. One plate is sufficient for three experiments.
Remove the lid and irradiate the cells. Once at 254 nanometers with 150 millijoules per centimeter squared, harvest the cells by scraping them off the plates with a cell lifter transfer two milliliters of cell suspension into each of three micro tubes to pellet the cells. Spin them at top speed for 10 seconds at four degrees Celsius.
Then remove the supinate snap, freeze the cell pellets on dry ice and store up minus 80 degrees Celsius until use. Add 100 microliters of protein, a dyna beads per experiment to a fresh micro tube. If working with mouse or goat antibodies, use protein G dyna beads.
Wash the beads twice with lysis buffer, which can be found in the accompanying written protocol. Resuspend the beads in 100 microliters of lysis Buffer with two to 10 micrograms of antibody. Rotate the tube at room temperature for 30 to 60 minutes.
Then wash the beads three times with 900 microliters of lysis buffer and leave them in the last wash until you are ready to proceed. To immunoprecipitation Thor cell palace on ice. Resus suspended in one milliliter of lysis buffer and transfer it to a 1.5 milliliter micro tube.
Prepare a one to 500 dilution of RNAs one. Then add 10 microliters together with two microliters of turbo DNAs to the cell.Lysate. Incubate the samples for exactly three minutes at 37 degrees Celsius, shaking at 1, 100 rotations per minute, immediately transfer to ice.
Then spin the samples at four degrees Celsius and 22, 000 times gravity for 20 minutes to clear the lysate carefully collect the SUP natant, leaving about 50 microliters of lysate with the pellet to immuno precipitate the beads. Remove the wash buffer, then add the prepared cell lysates. Rotate the samples for two hours at four degrees Celsius.
Discard the SUP agent and wash the beads twice with 900 microliters of high salt buffer, which can be found in the accompanying written protocol. Finally, wash the beads twice with 900 microliters of wash buffer. Then proceed to dephosphorylate and add a linker to the RNA three prime ends.
To label the RNA five prime ends working behind a plexiglass shield. Remove the supinate from the last wash of the linker ligation reaction and re suspend the beads in eight microliters of hot PNK mix. Incubate for five minutes at 37 degrees Celsius.
Remove the hot PNK mix and reus. Suspend the beads in 20 microliters of one times new page loading buffer. Then incubate the sample on a thermo mixer at 70 degrees Celsius for 10 minutes.
Immediately place the sample on a magnet to precipitate the empty beads. Load the samples onto a four to 12%new page. Biss gel according to the manufacturer's instructions.
Also, load five microliters of a prestained protein size marker. Run the gel for 50 minutes at 180 volts. Transfer the protein RNA complexes from the gel to a nitrocellulose membrane using the NOVA wet transfer apparatus according to the manufacturer's instructions.
After the transfer, rinse the membrane in PBS buffer. Then wrap it in Saran wrap and expose it to film at minus 80 degrees Celsius. Perform exposures for 30 minutes, one hour and overnight.
Next, isolate the membrane region according to the protein RNA complexes from the low RNAs. Experiment using the auto radiograph as a mask. Recover the RNA from the membrane with proteinase K digestion and phenol chloroform extraction.
Reverse transcribe the RNA using one of the AYP primers to gel purify the CDNA. Mix six microliters of CDNA sample with six microliters of two times TBE urea loading buffer. Just before loading the samples onto a gel.
Heat the samples to 80 degrees Celsius for three minutes. Then load the samples onto a precast 6%TBE urea gel along with a low molecular weight marker. Allow the gel to run for 40 minutes at 180 volts as described by the manufacturer using the upper dye and the marks on the plastic gel support to guide excision.
Cut three bands at 120 to 200 nucleotides 85 to 120 nucleotides and 70 to 85 nucleotides. Note that the arc lip primer and the L three sequence together account for 52 nucleotides of the clip sequence. Add 400 microliters of te and crush the gel slice into small pieces.
Using a one milliliter syringe plunger incubate the gel pieces with shaking at 1, 100 rotations per minute for two hours at 37 degrees Celsius. Place two one centimeter glass pre-filters into a CoStar spinx column. Transfer the liquid portion of the sample to the column spin for one minute at 13, 000 rotations per minute into a 1.5 milliliter tube.
Add 0.5 microliters of glyco blue and 40 microliters of sodium acetate pH 5.5. Then mix the sample. Add one milliliter of 100%ethanol.
Mix again and precipitate the sample overnight at minus 20 degrees Celsius. Finally, circularize the CD NA molecules to attach the adaptor region to the five prime end. Next re linearize and PCR amplify according to the accompanying written protocol prior to the sequencing of the eye clip library, the success of the experiment can be monitored at two steps.
The autoradio graph of the protein RNA complex after membrane transfer and the gel image of the PCR products in the auto radiograph of the low RNA samples, diffuse radioactivity should be seen above the molecular weight of the protein for high RNA samples. This radioactivity is focused closer to the molecular weight of the protein. When no antibody is used in the immunoprecipitation, no signal should be detected.
Further important controls for specificity of the immunoprecipitation either emits UVE irradiation or use cells that do not express the protein of interest. The gel image of the PCR products should demonstrate a size range that corresponds to the CD NA fraction. Note that the PCR primers, P three Celexa and P five Celexa introduce an additional 76 nucleotides to the size of the CD NA.If no antibody is used during the immunoprecipitation, no corresponding PCR product should be detected.
While attempting this procedure, it is important to remember that each of the 64 steps is critical and has to be performed with a hundred percent accuracy. ICL data can be computationally integrated with transcriptum wide functional assays. This can, for example, gain insights into the position dependent regulation of RNA processing.
For computational data analysis, we would like to refer to our recent publications in collaboration with Nicholas KA European Bioinformatics Institute and bla zupan at the University of Liana. And now it's time that you start your own experiments. Have fun.
