BrU-labeling and immunoprecipitation of newly synthesized RNA

BrU-labeling and immunoprecipitation of newly synthesized RNA Anne Kruse Hollensen1, Christian Kroun Damgaard11Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark ProtocolSeeding of cellsSeed cells in eight p6 dishes per series of samples6 ml cell culture medium/dish PulseAspirate 4 ml of medium from each plate and pool medium from each series of samples. For each series of samples:For one dish (–BrU): Aspirate the remaining medium from the cells and add 3 ml of the collected medium.For the remaining four dishes (+BrU): Add 0.25 M 5-bromouridine (BrU) to a final concentration of 2 mMAspirate the remaining medium from the cells Add 3 ml +BrU medium to each plate Incubate for 1 hour ChaseAspirate medium Add 4 ml medium to the cells and aspirate Add 4 ml medium to the cells Incubate at 37˚ C for 5 min Aspirate Add 4 ml medium Incubate at 37˚ C Harvest the –BrU samples after 35 minutes Harvest the 0 h +BrU samples after 50 minutes Harvest the remaining samples after additionally 3 h, 6 h, and 9 h Harvest cellsPlace cell-plates on ice Carefully wash each plate once in cold PBS Lyse the cells in 1 ml cold Trizol Transfer the lysate to an Eppendorf tube Store at -20˚ C until purification of RNA RNA purificationPurify RNA according to Trizol protocol Resuspend RNA in nuclease free H2O Measure concentrations Dilute to 1 µg/µl   Immunoprecipitation of BrU-labeled RNAReagents:Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen)1xBrU-IP Buffer w. 1 mg/ml Heparin (see recipe below)Heparin (1 mg/ml)a-BrdU antibody, clone 3D4 (555627, BD Pharmingen)1xBrU-IP Buffer (see recipe below)BSA (100 mg/ml)0.25 M 5-bromouridine (5-BrU)BrU-labeled total RNA at 1 mg/mlRiboLock (40 U/µl)Phenol:Chloroform pH 6.6Chloroform3M NaAcGlycogen (10 µg/µl) Equipment:RotatorMagnetic standTable-top centrifugeThermo-shaker at 80˚ CCold centrifugeDry iceIce trayBuffers:Use RNase-free H2O for all buffers 2xBrU-IP buffer: 40 mM Tris-HCl pH 7.5500 mM NaCl 2xBrU-IP buffer + BSA/RiboLock:2x BrU-IP buffer1 µg/µl BSA80 U/ml RiboLock 1xBrU-IP buffer + Heparin:1xBrU-IP buffer1 mg/ml Heparin 1xBrU-IP buffer:1xBrU-IP buffer0.5 µg/µl BSA20 U/ml RiboLockElution buffer:0.1 % SDS Prepare beads/antibody:Beads/antibody are prepared in batch followed by aliquoting (beads: 20 µl/sample, antibody: 2.5 µl/sample)Resuspend the Goat anti-mouse IgG dynabeads thoroughly in the vial to obtain a uniform brown suspensionTransfer the desired volume of Dynabeads to an Eppendorf tube Spin briefly in table-centrifugePlace the tube on a magnet for 1-2 min Remove supernatant Remove the tube from the magnetWash beads (add 400 µL 1xBrU-IP buffer  mix by pipetting carefully a couple of times  rotate 2 minutes at room temperature  spin briefly in table-centrifuge  place the tube on a magnet for 1-2 min  remove supernatant) Repeat the wash Resuspend the beads in 1 ml 1xBrU-IP buffer with Heparin Rotate 30 min at room temperatureWash beads in 1xBrU-IP bufferResuspend the beads in 1 ml 1xBrU-IP bufferMix by pipetting carefully a couple of timesAdd the desired volume of a-BrdU antibody to the mixtureWash beads three times in 1xBrU-IP buffer Resuspend beads in 50 µl 1xBrU-IP buffer/sample Add 0.25 M 5-BrU to a final concentration of 1 mM BrURotate 30 minutes at room temperatureWash beads three times in 1xBrU-IP bufferResuspend beads in 50 µl 1xBrU-IP buffer/sample Prepare RNA samples:Transfer 40 µg (1 µg/µl) RNA for each sample to new Eppendorf tubes Add 160 µl H2ODenature the RNA by incubating at 80˚ C for 2 min Spin down briefly in table-centrifuge Add 200 µl 2xBrU-IP buffer with BSA/RiboLock Bind RNA:Add 50 µl resuspended prepared beads to each RNA sample Rotate 1 h at room temperature Spin down briefly in table-centrifugePlace the tube on a magnet for 1-2 min Carefully remove supernatant and transfer to Eppendorf tubes (unbound RNA, store at -20˚ C)Wash beads four times in 1xBrU-IP bufferResuspend the beads in 200 µl Elution buffer and proceed quickly to “Elute bound RNA” Elute bound RNA:Add 200 µl phenol/chloroform pH 6.6 to the resuspended beads Vortex Spin for 3 min at full speed at 4˚ C Transfer the supernatant (190 µl) to a tube containing 200 µl chloroformVortex Spin for 3 min at full speed at 4˚ C Transfer supernatant (180 µl) to a tube containing 18 µl 3M NaAc pH 6.0 and 2 µl glycogen (10 µg/µl) Add 500 µl 96% EtOH Mix by inverting the tube a couple of times Place on dry ice for 15 min or at -20˚ C for >1 hour Spin at full speed at 4˚ C for >30 min Discard supernatant completely without disturbing the pellet Wash with 185 µl 75% EtOH Spin at full speed at 4˚ C for 5 min Discard supernatant completely without disturbing the pellet Dry the RNA pellet Resuspend the RNA in 10 µl nuclease free H2O ReferencesHollensen, A.K., Thomsen, H.S., Lloret-Llinares, M., Kamstrup, A.B., Jensen, J.M., Luckmann, M., Birkmose, N., Palmfeldt, J., Jensen, T.H., Hansen, T.B., and Damgaard, C.K. (2020). circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation. Elife 9. 10.7554/eLife.58478. Kofoed, R.H., Betzer, C., Lykke-Andersen, S., Molska, E., and Jensen, P.H. (2018). Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation. J Vis Exp. 10.3791/57056.