The overall goal of this procedure is to determine the transcriptome wide binding sites of RNA binding proteins or RVPs. This is accomplished by first using photoreactive nucleoside analogs for in vivo labeling of nascent transcripts. The second step of the procedure is to cross-link RNA and RNA binding proteins by 365 nanometer UV light.
The third step of the procedure is to immuno precipitate the investigated RNA binding protein and to recover the cross-link to RNA. The final step of the procedure is to convert the RNA into a CD NA library and deep sequence. It ultimately results can be obtained that show transcriptome wide binding sites of the RVPs and enable the definition of the RNA recognition element and its binding preferences.
Hi, my name is Marcus Hoffner from the lab of Tom SEL at the Rockefeller University in New York. Today we'll be showing a method called par lip or photoable rib nucleoside analog enhanced cross-linking and immunoprecipitation. We use this procedure in our lab to identify on a transcriptome by its scale binding sites of RNA binding proteins and their binding motifs and preferences.
Okay, so let's get started. In vivo UV cross-linking begins in a laminar flow hood. In this protocol we will use tagged IGF two BP one cells as an exemplary model using sterile technique.
Expand HEK 2 93 cells stably expressing flag HA tagged IGF two BP one under control of a Tet repressor in HEK 2 93 cell culture medium use between 100 and 400 times 10 to the sixth cells and grow them to approximately 80%co fluency. At 14 hours before cross-linking the cells had the photoreactive riboside analog for thio uridine or for SU directly to the cell culture medium. Alternatively, six th guine or six SG can be used following addition of the photoreactive riboside induce expression of the flag HA tagged IGF two BP one by the addition of doxycycline cells are then incubated overnight in cell incubator.
Next, decant the growth medium. Then wash the cells once with 10 milliliters of ice cold phosphate buffered saline per plate. Then remove the PBS completely by decanting.
Place the uncovered plates on a tray with ice and irradiate them with 365 nanometer UV light in a straddle linker 2, 400 UV crosslinker or equivalent subsequent to UV radiation, scrape the cells off with a rubber policeman in one milliliter of PBS per plate. Transfer the cells to 50 milliliter centrifugation tubes and centrifuge at 500 G for five minutes at four degrees Celsius. Discard the super name and retain the cell pellet.
At this point, cell pellets can be flash frozen in liquid nitrogen and stored at minus 80 for at least 12 months. To continue with the procedure reus suspend the pellet in three volumes of one X NP 40 lysis buffer, and incubate on ice for 10 minutes. Clear the cell lysate by centrifugation at 13, 000 G for 15 minutes at four degrees Celsius.
Following centrifugation, clear the lysate further by filtering it through a five micron membrane syringe filter. Finally, add RNAs T one to a final concentration of one unit per microliter and incubate in a water bath at 22 degrees Celsius for 15 minutes. Subsequently, cool the reaction on ice for five minutes.
Following in vivo UV cross-linking. Prepare the magnetic beads for immunoprecipitation. Transfer 10 microliters of dyna beads protein G magnetic particles per one mil of cell lysate to a 1.5 milliliter micro centrifuge tube.
Then place the tube containing the beads on a magnetic stand for one to two minutes. Remove the supernatant and add one milliliter of citrate phosphate buffer to the tube while it is on the magnetic separator. Next, cap the tube and vortex the sample to resuspend the beads.
Centrifuge briefly to collect any beads remaining in the tube cap. Repeat this wash with an additional one milliliter of citrate phosphate buffer. Following the wash resus, suspend the beads and citrate phosphate buffer at a volume of two times the original volume of beet suspension.
Then add 0.25 milligrams of Anti-Flag M two monoclonal antibody per one milliliter of beat suspension. Incubate the suspension on a rotating wheel for 40 minutes at room temperature after the incubation, wash the beads twice in one milliliter of citrate phosphate buffer as previously described, this wash serves to remove any unbound antibody following the wash Resus, suspend the beads and citrate phosphate buffer as before four. To begin immunoprecipitation, add the freshly prepared antibody conjugated magnetic beads to the RNAs T one treated cell lysate.
Transfer the mixture to 15 milliliter centrifugation tubes and rotate for one hour at four degrees Celsius. Following incubation, collect the magnetic beads, discard the supernatant resuspend beads in one milliliter of IP wash buffer and transfer them to 1.5 milliliter centrifuge tubes. Wash the beads as described before two times in one milliliter of IP wash buffer.
After the final wash, resus suspend the beads in the original bead volume of IP wash buffer. Then add RNAT one to a final concentration of 100 units per microliter. Incubate the bead suspension in a 22 degree Celsius water bath for 15 minutes.
Subsequently cool the suspension on ice for five minutes. Next, wash the beads three times in one milliliter of high salt wash buffer. Then resuspend the beads in one original bead volume of dephosphorylation buffer.
Add calf intestinal alkaline phosphatase to a final concentration of 0.5 units per microliter and incubate the suspension for 10 minutes at 37 degrees Celsius. Wash the beads twice in one milliliter of phosphatase wash buffer and then twice more in polynucleotide kinase or PNK buffer without DTT. Finally resuspend the beads in one original bead volume of PNK buffer.
To begin radio labeling of cross-linked RNA. Add gamma 32 PATP and T four PNK to one original bead volume of the freshly prepared bead suspension. Incubate the suspension for 30 minutes at 37 degrees Celsius.
Next, add non-radioactive a TP to the suspension and incubate it for another five minutes at 37 degrees Celsius. Then wash the magnetic beads five times with 800 microliters of PNK buffer without DTT. Finally, resuspend the beads in 70 microliters of SDS page.
Loading buffer incubate the radio label suspension for five minutes in a heat block at 95 degrees Celsius to denature and release the immuno precipitated RNA binding proteins with the cross-linked RNA. Then remove the magnetic beads on the separator and transfer the super natin to a clean 1.5 milliliter micro centrifuge tube Load 40 microliters of the suade per well of a novox bis triss four to 12%precast poly acrylamide gel and run the gel for 55 minutes at 200 volts. Once the gel has run, disassemble the gel chamber and dismantle the plates, leaving the gel mounted on one plate to facilitate the alignment of the gel to the phospho imager paper printout.
Implant three tiny radioactive gel pieces asymmetrically at the gel corners. Wrap the gel in a plastic film such as Saran wrap to avoid contamination. Expose the gel to a blank phospho imager screen for one hour and it on a phospho imager.
Next, align the gel on top of the phospho imager printout using the implanted gel pieces for orientation. Cut out the bands that correspond to the expected size of the RNA binding protein or RBP. Transfer the bands to a dtu dialyzer MIDI tube and add 800 microliters of one XSDS running buffer electro elute.
The cross-linked R-N-A-R-B-P complex at 100 volts for two hours per the manufacturer's instructions. Subsequent to the electro elution. Add proteinase K buffer and proteinase K incubate the solution for 30 minutes at 55 degrees Celsius.
Recover the RNA by acidic phenol chloroform isoamyl alcohol extraction, followed by a chloroform extraction. Then add one microliter of glycogen and precipitate the RNA by adding three volumes of ethanol. Finally dissolve the resulting pellet in 10.5 microliters of water.
Once the RNA has been recovered, carry it through a standard CDNA library preparation protocol and perform careful bioinformatic analysis of the sequence reads. Representative results for a par clip performed with HK 2 93 cells expressing flag H ha tagged IGF two BP one are shown with the photoreactive nucleoside analogs four SU and six SG cross-linking efficiency of the alternative photoreactive riboside six SG is generally lower than for four su. The lower cross-linking efficiency might result in a higher background of sequences derived from fragments of abundant cellular RNAs and therefore more cells should be initially used for the experiment.
Different par clip photoreactive uridine analogs are compared to traditional UV 2 54 nanometer cross-linking or clip as seen here. UV 2 54 nanometer RNA protein cross-linking has limited cross-linking efficiency. The intensity of the appropriately sized radioactive band indicates whether the par clip experiment has worked and whether sufficient RNA has been isolated to carry through a small RNA sequencing protocol.
The frequencies of characteristic mutations in the sequenced reads following bioinformatic analysis are indicative of successfully cross-linked sequences. These mutations are T two C transitions when using four SU and G two A transitions when using success. G.The defining feature of park lip is the possibility to derive the location of the cross link by scoring for a characteristic mutation residing in the CD NA libraries prepared from the recovered RNAs.
This allows for the easy separation of the signal derived from specifically bound RNAs, from the noise of sequences derived from co isolated fragments of abundant cellular RNAs. So we've shown you today how to isolate RNA segments, specifically recognized by RNA binding proteins. When doing this procedure.
It is important to work RNAs free. So that was it. Thanks for watching and good luck with the experiments.
