ChIP protocol Chromatin fragmentation using the Covaris S2 sonicator by Ethan Ford (version 12/1/11) X- link Cells

Transcription

1 ChIP protocol Chromatin fragmentation using the Covaris S2 sonicator by Ethan Ford (version 12/1/11) X- link Cells 1. Grow six 15 cm plates of HeLa cells to 90% confluency. 2. Remove media and add wash with 10 ml of PBS. 3. Add 10 ml Fixing Buffer with 1% formaldehyde (see note #1 and #2). 4. Incubate 5 min at room temp on rocking platform. 5. Add 0.5 ml 2.5 M glycine to each plate (final concentration M). 6. Incubate 5 min at room temp on rocking platform. 7. Pour off media and rinse 2x with PBS. 8. Harvest cells with 10 ml PBS using rubber policeman into a 15 ml tube. 9. Rinse plates with an additional 5 ml of PBS to get remaining cells and transfer to the same 15 ml tube. 10. Spin cells down at 3k rpm for 5 min. Prepare Chromatin 11. Remove supernatant and resuspend cells in 15 ml Lysis Buffer. 12. Incubate 10 min on ice. 13. Spin cells down at 4k rpm for 5 min. 14. Remove supernatant and resuspend cells in 10 ml Wash Buffer 15. Spin cells down at 4k rpm for 5 min. 16. Repeat steps 14 and 15 one more time. 17. Carefully remove supernatant as to not disturb the pellet. 18. Carefully add 5 ml Shearing Buffer and very gently turn tube sideways and twist to wash all the Wash Buffer off from the side of the tube while leaving the pellet completely intact. 19. Spin cells down at 4k rpm for 5 min. 20. Repeat steps 18 and 19 one more time. 21. Resuspend pellet in 8 ml Shearing Buffer 22. Transfer sample to Covaris TC12x12mm Tubes with AFA Fiber being sure to fill tubes completely (a little more then 1 ml per tube - see note #3). 23. Sonicate with a Covaris S2 for with the following settings: Time 12 min (see note #4) Duty Cycle 5% Intensity 4 Cycles per Burst 200 Temperature 4 C Power mode Frequency Sweeping Degassing mode Continuous AFA Intensifier Water Level No Intensifier 8 (water level should be 1 mm below TC12x12 tube cap)

2 24. Remove samples from TC12 tubes and transfer to new 15 ml tube. 25. Repeat sonication with the rest of the samples and combine. 26. Add 10% Triton X- 100 and 5M NaCl so that the final concentration of the sample is 1% Triton X- 100 and 150 mm NaCl, i.e. to 8.70 ml of chromatin add 1 ml of 10% Triton X- 100 and 300 μl of 5 M NaCl. 27. Mix and transfer to 8 microfuge tubes. 28. Spin chromatin in microfuge for 10 min. 29. Combine supernatants in a new 15 ml tube. 30. Filter chromatin through 2 μm syringe filter. 31. Remove syringe from filter, remove plunger, reattach syringe and pass an additional 1 ml of IP Buffer through syringe. Immunoprecipitation 32. Take 100 μl of chromatin and put aside for input control. 33. Use 1 ml of chromatin per I.P. (extra chromatin can be stored at - 80 C). 34. Add approximately μg affinity purified antibody, 1-2 μg purified IgG, or 1-4 ul crude serum. 35. Incubate at 4 C overnight. 36. Equilibrate beads. Prepare one 1.5 ml tube for each for each immunoprecipitatin with 1 ml Shearing Buffer and desired amount of Protein G- Dyna beads to each tube. Mix by inverting and spin at 3k rpm in microfuge briefly to get liquid off top of tube. Place in magnetic rack and remove all liquid. Note: Protein- G beads are a significant source of background so it is best to use as few as possible. The stated binding capacity is 200 ng antibody/μl. 37. Add the chromatin from each I.P. to the appropriate tube with equilibrated beads. 38. Incubate at 4 C for 1.5 hours in tube rotator. 39. Spin briefly in microfuge at 3k RPM. Place in magnetic rack and remove liquid. 40. Use filter tips for all remaining steps. 41. Wash 2 times with Low Salt Wash Buffer. Wash instructions: Add 1 ml Wash Buffer, mix by inverting or pipeting. If mixed by inverting, spin briefly to get liquid off the top of the tube. Place in magnetic rack and remove all liquid. Transfer beads to a new tube every other wash. 42. Wash 2 times with High Salt Wash Buffer 43. Wash 1 times with LiCl Wash Buffer 44. Wash 1 time with 1ml TE ph To elute chromatin (ChIP ed DNA), add 49 μl Proteinase K Digestion Buffer and 1 μl Proteinase K (20 mg/ml). To your Input DNA add 2 μl 10% SDS and 1 μl Proteinase K (20 mg/ml). 46. Incubate at 50 C for 15 min with occasional vortexing. 47. Place ChIP ed DNA in magnetic rack for 2 minutes. 48. Transfer supernatant to new tube. 49. To ChIP ed and Input DNA add 3 μl 5M NaCl and 0.5 μl 30 mg/ml RNase A.

3 50. Reverse X- linking by incubating 4 hours to overnight at 65 C in hybridization oven to prevent condensation on top of tube. 51. Add 1.5 μl 20 mg/ml Proteinase K. 52. Incubate 1 hour at 50 C. Vortex a couple times during incubation. Purify DNA with AMPure Beads (See note #5) 53. Add 50 μl of well- mixed Ampure XP beads and 60 μl 20 % PEG M NaCl 54. Mix by pipetting up and down several times (See Note 9). 55. Incubate at room temperature for 15 min. 56. Place on magnetic rack for 5 min. 57. Remove and discard the supernatant. When removing supernatant do so very slowly with pipetman being careful not to take any beads. 58. Keep sample in magnetic rack and add 900 μl of freshly prepared 80% ethanol. 59. Incubate for 30 seconds. Remove and discard ALL the supernatant. 60. Repeat steps 58 and 59 one more time. 61. Let the beads dry at room temperature for 2 minutes. 62. Add 45.5 μl TE/10 and pipet up and down several times until pellet beads are completely resuspended. 63. Incubate at room temperature for 2 minutes. 64. Place in magnetic rack for 5 minutes. 65. Transfer 44 μl of the supernatant to a 0.2 ml PCR tube. Post- ChIP analysis 66. Use 4 μl of eluted DNA for with QuantIT HS DNA Assay Kit (Invotrogen) 67. Run 5 μl of input DNA on a 2.2% agarose gel. 68. Use 2.5 μl DNA for qpcr. 69. Use 10 ng for ChIP- seq library preparation. Notes: 1) It is highly recommended to use formaldehyde from 16% single use ampules or freshly prepared 16% formaldehyde from paraformaldehyde (see protocol for preparing 16% formaldehyde). 2) It is highly recommended that you try a time course of crosslinking. Different proteins crosslink at different efficiencies to the chromatin. It is also possible with some proteins to increase the efficiency of your ChIP by using a second crosslinker such as DSG, EGS or DMA. 3) It is critical to use these specific tubes from Covaris with the appropriate tube holder. It is also critical that the tubes are filled completely. If your sample does not fill the tube add extra Shearing Buffer to the sample until the tube is full.

4 4) To optimize chromatin fragmentation, during sonication remove 25 μl aliquots of chromatin at various time points. A good start is 2, 5, 10 and 15 min. Each time adding an additional 25 μl of shearing buffer to sample to keep tube full. Add 75 μl of H2O to chromatin aliquot and proceed with reverse X- linking, Proteinase K treatment and purify with Qiagen MinElute column according to manufactures instructions. Elute with 30 μl elution buffer and run 3 μl on a 2.2% agarose gel. It may also be helpful to assess the integrity of your epitope during the shearing process. To do so, remove 5 μl of your sample at each time point. Add 25 μl of H20 and 2 μl 5M NaCl. Incubate overnight in hybridization oven or PCR machine at 65 C. Run 10 μl on a SDS- PAGE gel and Western Blot with an antibody to your protein. 5) AMPure XP guidelines: a) Make sure that you achieve a homogenous solution of the beads and PEG by pipetting up and down a sufficient number of times. b) When removing the supernatant after the binding step, I remove all but 5 μl with a P200 pipetman and then go back a carefully remove the last 5 μl with a P20 pipetman unless I have a lot of samples to process, then I just remove it all in one step with the P200. c) When washing with ethanol the beads stick to the wall of the tube better so you can remove the supernatant more quickly and less carefully. However, I make sure I remove all traces of ethanol by going through each tube a second time. d) At the elution step, when transferring the eluted DNA to a new tube it is important not to accidentally carryover any beads. I carefully look inside the tube as I am pipetting up the eluted DNA to make sure I don t accidentally take up and beads. Then I look at the eluted DNA in the pipet tip see if I can see any beads. If there are, simply pipet the breads back into the tube you took them from and wait a few minutes for the sample to separate again in the magnetic rack. At this step it is important to leave at lease 1.5 μl behind as it is not possible to remove all the liquid and not take any beads. 6) This protocol can easily be scaled up or down. The number of cells per ml of shearing buffer should not exceed 5x10 6 according to Covaris. Fixing Buffer 50 mm Hepes- KOH, ph mm NaCl 1 mm EDTA, ph mm EGTA, ph 8.0 Fixing Buffer with 1% Formaldehyde ml Fixing Buffer 625 μl 16% formaldehyde note: this solution must be prepared fresh and used immediately

5 Lysis Buffer 50 mm HEPES ph mm NaCl 1 mm EDTA 10% glycerol 0.5% NP % Triton X- 100 Wash Buffer 10 mm Tris- Cl, ph mm NaCl 1 mm EDTA, ph mm EGTA, ph 8.0 Shearing Buffer 0.1% SDS 1 mm EDTA 10 mm Tris, ph 8.1 IP Buffer 0.1% SDS 1 mm EDTA 10 mm Tris, ph 8.1 1% Triton X mm NaCl Low Salt Wash Buffer 0.1% SDS 1% Triton X mm EDTA 20 mm Hepes- KOH, ph mm NaCl High Salt Wash Buffer 0.1% SDS 1% Trtiton X mm EDTA 20 mm Hepes- KOH, ph mm NaCl LiCl Wash Buffer 100 mm Tris- HCl, ph M LiCl 1% NP- 40 1% Sodium Deoxycholate

6 Proteinase K Digestion Buffer 20 mm HEPES, ph mm EDTA 0.5% SDS TE/10 10 mm Tris- HCl, ph mm EDTA

7 Figure 1. Time course of shearing time course for HeLa chromatin. Six 15 cm HeLa plates were grown to confluence and cross- linked for 5 min with 1% formaldehyde according to protocol. The final nuclear pellet was resuspended in 10 ml of shearing buffer. 1 ml was sheared under the perameters described above. 25 μl was removed at each time point and processed as described in Note 3 (above).

8 Figure 2. Western blot of a chromatin bound protein after Covaris shearing and reverse cross- linking.