BrU-labeling and immunoprecipitation of newly synthesized RNA

BrU-labeling and immunoprecipitation of newly synthesized RNA

Anne Kruse Hollensen¹, Christian Kroun Damgaard¹

¹Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

Protocol

Seeding of cells

Seed cells in eight p6 dishes per series of samples

6 ml cell culture medium/dish

Pulse

Aspirate 4 ml of medium from each plate and pool medium from each series of samples.

For each series of samples:

For one dish (–BrU): 

1. Aspirate the remaining medium from the cells and add 3 ml of the collected medium.

For the remaining four dishes (+BrU): 

1. Add 0.25 M 5-bromouridine (BrU) to a final concentration of 2 mM

2. Aspirate the remaining medium from the cells 

3. Add 3 ml +BrU medium to each plate 

4. Incubate for 1 hour

Chase

1. Aspirate medium 

2. Add 4 ml medium to the cells and aspirate 

3. Add 4 ml medium to the cells 

4. Incubate at 37˚ C for 5 min 

5. Aspirate 

6. Add 4 ml medium 

7. Incubate at 37˚ C 

8. Harvest the –BrU samples after 35 minutes 

9. Harvest the 0 h +BrU samples after 50 minutes 

10. Harvest the remaining samples after additionally 3 h, 6 h, and 9 h

Harvest cells

1. Place cell-plates on ice 

2. Carefully wash each plate once in cold PBS 

3. Lyse the cells in 1 ml cold Trizol 

4. Transfer the lysate to an Eppendorf tube 

5. Store at -20˚ C until purification of RNA

RNA purification

1. Purify RNA according to Trizol protocol 

2. Resuspend RNA in nuclease free H₂O 

3. Measure concentrations 

4. Dilute to 1 µg/µl

Immunoprecipitation of BrU-labeled RNA

Reagents:

1. Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen)

2. 1xBrU-IP Buffer w. 1 mg/ml Heparin (see recipe below)

3. Heparin (1 mg/ml)

4. a-BrdU antibody, clone 3D4 (555627, BD Pharmingen)

5. 1xBrU-IP Buffer (see recipe below)

6. BSA (100 mg/ml)

7. 0.25 M 5-bromouridine (5-BrU)

8. BrU-labeled total RNA at 1 mg/ml

9. RiboLock (40 U/µl)

10. Phenol:Chloroform pH 6.6

11. Chloroform

12. 3M NaAc

13. Glycogen (10 µg/µl)

Equipment:

1. Rotator

2. Magnetic stand

3. Table-top centrifuge

4. Thermo-shaker at 80˚ C

5. Cold centrifuge

6. Dry ice

7. Ice tray

Buffers:

Use RNase-free H₂O for all buffers

2xBrU-IP buffer: 

40 mM Tris-HCl pH 7.5

500 mM NaCl

2xBrU-IP buffer + BSA/RiboLock:

2x BrU-IP buffer

1 µg/µl BSA

80 U/ml RiboLock

1xBrU-IP buffer + Heparin:

1xBrU-IP buffer

1 mg/ml Heparin

1xBrU-IP buffer:

1xBrU-IP buffer

0.5 µg/µl BSA

20 U/ml RiboLock

Elution buffer:

0.1 % SDS

Prepare beads/antibody:

1. Beads/antibody are prepared in batch followed by aliquoting (beads: 20 µl/sample, antibody: 2.5 µl/sample)

2. Resuspend the Goat anti-mouse IgG dynabeads thoroughly in the vial to obtain a uniform brown suspension

3. Transfer the desired volume of Dynabeads to an Eppendorf tube 

4. Spin briefly in table-centrifuge

5. Place the tube on a magnet for 1-2 min 

6. Remove supernatant 

7. Remove the tube from the magnet

8. Wash 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) 

9. Repeat the wash 

10. Resuspend the beads in 1 ml 1xBrU-IP buffer with Heparin 

11. Rotate 30 min at room temperature

12. Wash beads in 1xBrU-IP buffer

13. Resuspend the beads in 1 ml 1xBrU-IP buffer

14. Mix by pipetting carefully a couple of times

15. Add the desired volume of a-BrdU antibody to the mixture

16. Wash beads three times in 1xBrU-IP buffer 

17. Resuspend beads in 50 µl 1xBrU-IP buffer/sample 

18. Add 0.25 M 5-BrU to a final concentration of 1 mM BrU

19. Rotate 30 minutes at room temperature

20. Wash beads three times in 1xBrU-IP buffer

21. Resuspend beads in 50 µl 1xBrU-IP buffer/sample

Prepare RNA samples:

1. Transfer 40 µg (1 µg/µl) RNA for each sample to new Eppendorf tubes 

2. Add 160 µl H₂O

3. Denature the RNA by incubating at 80˚ C for 2 min 

4. Spin down briefly in table-centrifuge 

5. Add 200 µl 2xBrU-IP buffer with BSA/RiboLock

Bind RNA:

1. Add 50 µl resuspended prepared beads to each RNA sample 

2. Rotate 1 h at room temperature 

3. Spin down briefly in table-centrifuge

4. Place the tube on a magnet for 1-2 min 

5. Carefully remove supernatant and transfer to Eppendorf tubes (unbound RNA, store at -20˚ C)

6. Wash beads four times in 1xBrU-IP buffer

7. Resuspend the beads in 200 µl Elution buffer and proceed quickly to “Elute bound RNA”

Elute bound RNA:

1. Add 200 µl phenol/chloroform pH 6.6 to the resuspended beads 

2. Vortex 

3. Spin for 3 min at full speed at 4˚ C 

4. Transfer the supernatant (190 µl) to a tube containing 200 µl chloroform

5. Vortex 

6. Spin for 3 min at full speed at 4˚ C 

7. Transfer supernatant (180 µl) to a tube containing 18 µl 3M NaAc pH 6.0 and 2 µl glycogen (10 µg/µl) 

8. Add 500 µl 96% EtOH 

9. Mix by inverting the tube a couple of times 

10. Place on dry ice for 15 min or at -20˚ C for >1 hour 

11. Spin at full speed at 4˚ C for >30 min 

12. Discard supernatant completely without disturbing the pellet 

13. Wash with 185 µl 75% EtOH 

14. Spin at full speed at 4˚ C for 5 min 

15. Discard supernatant completely without disturbing the pellet 

16. Dry the RNA pellet 

17. Resuspend the RNA in 10 µl nuclease free H₂O

References

Hollensen, 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.