ChIP Sonication 1%SDS - Ethan-omics | Everything …€™sbest%touseasfew%aspossible.%Thestated...
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Transcript of ChIP Sonication 1%SDS - Ethan-omics | Everything …€™sbest%touseasfew%aspossible.%Thestated...
ChIP protocol Chromatin fragmentation by sonication in 1% SDS by Ethan Ford (version 7/4/11) X-‐link Cells
1. Grow two 15 cm plates of HeLa cells to 90% confluency. 2. To each 15 cm plate of HeLa cells add 685 μl of 36.5% formaldehyde (final
concentration 1%) 3. Incubate 10 min at room temp on rocking platform. 4. Add 1.25 ml 2.5 M glycine to each plate (final concentration 0.125 M). 5. Incubate 5 min at room temp on rocking platform. 6. Pour off media and rinse 3x with PBS. 7. Harvest cells with 10 ml of PBS using rubber policeman into a 15 ml tube. 8. Rinse plates with an additional 5 ml of PBS to get remaining cells and
transfer to the same 15 ml tube. 9. Spin cells down at 2.5k rpm for 5 min.
Prepare Chromatin
10. Remove supernatant and resuspend cells in 15 ml ‘Cell Lysis Buffer’. 11. Incubate 15 min on ice. 12. (optional) Dounce homogenize with 15 strokes using A (tight) pestle. 13. Spin cells down at 2.5k rpm for 5 min. 14. Remove supernatant and resuspend cells in 1 ml ‘Nuclei Lysis Buffer’. 15. Sonicate Chromatin to an average length of <300 bp for ChIP-‐seq and <500
bp for ChIP-‐PCR. This needs to be optimized for each sonicator and cell type. It is vital that chromatin stays cold during sonication, thus you must sonicate in short pulses of no more then 10 seconds followed by cooling in an ice-‐water bath. See note at end of protocol.
16. Spin chromatin in microfuge for 10 min. 17. Transfer supernatant to new tube. 18. Add 9 ml of ‘IP Dilution Buffer’ (final SDS concentration must to be reduced
to 0.1%). 19. Filter chromatin through 2 μm syringe filter. 20. Remove syringe from filter, remove plunger, reattach syringe and pass an
additional 1 ml of ‘Chromatin Buffer’ through syringe.
Immunoprecipitation
21. Take 150 μl of chromatin and put aside for input control. 22. Use 1 ml of chromatin per I.P. (extra chromatin can be stored at -‐80˚ C). 23. Add approximately 250 ng affinity purified antibody, 1-‐2 μg purified IgG, or
1-‐4 ul serum. 24. Incubate at 4° C overnight. 25. Equilibrate beads. Prepare one 1.5 ml tube for each for each
immunoprecipitatin with 1 ml ‘Chromatin Buffer’ and desired amount of Protein G-‐Dyna beads to each tube. Note: protein-‐G beads are a significant
source of background so it’s best to use as few as possible. The stated binding capacity is 200 ng antibody/μl. 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.
26. Add the chromatin from each I.P. to the appropriate tube with equilibrated beads.
27. Incubate at 4˚ C for 1.5 hours in tube rotator. 28. Spin briefly in microfuge at 3k RPM. Place in magnetic rack and remove
liquid. 29. Use filter tips for all remaining steps. 30. 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.
31. Wash 2 times with ‘High Salt Wash Buffer’ 32. Wash 2 times with ‘LiCl Wash Buffer’ 33. Wash 1 time with 1ml TE pH 8.1. 34. To elute chromatin, add 50 μl 2% SDS, 0.1M NaHCO3 (make fresh) and
incubate 45 min at 65˚ C. Vortex every 10 min or so. 35. Spin briefly at 3k rpm and collect eluted DNA by placing in magnetic rack. 36. Collect remaining DNA by resuspending beads in 50 μl 0.1 M NaHCO3, vortex
briefly, spin briefly, place in magnetic rack, collect supernatant and combine with previous elution.
Reverse X-‐link
37. To ChIP’ed DNA add 6 μl 5M NaCl and 0.5 μl 30 mg/ml RNase A. To input DNA add 9 μl 3M NaCl and 0.5 μl 30 mg/ml RNase A.
38. Reverse X-‐linking by incubating 4 hours to overnight at 65˚ C. 39. To ChIP’ed DNA add 100 μl H2O, 4 μl 1M Tris-‐HCl pH 6.5 and 1.5 μl 20 mg/ml
Proteinase K. To input DNA add 150 μl H2O, 4 μl 1M Tris-‐HCl pH 6.5, 12 μl 10% SDS and 1.5 μl 20 mg/ml Proteinase K.
40. Incubate 2 hours at 50˚ C. Vortex a couple times during incubation. 41. Remove 100 μl of input DNA and discard.
Purify DNA with Qiagen MinElute columns
42. Add 10 μl 3M NaOAc, pH 5.2 and 1ml Qiagen Buffer PB. Mix by pipeting. 43. Apply 650 μl to Qiagen Minelute column. Spin for 10 sec in microfuge. 44. Remove all liquid from collection tube. 45. Apply remaining DNA to column. Spin 1 min at max speed. 46. Remove all liquid from collection tube. 47. Wash column by adding 750 μl Qiagen buffer PE to column 48. Let sit for 2 min. Spin 1 min at max speed 49. Remove all liquid from collection tube. 50. Spin an additional 2 min to dry column.
51. Place column in new 1.5 ml tube and elute DNA by adding 40 μl Elution Buffer to column, let sit for 5 min and spin 1 min.
52. Transfer eluted DNA to new 1.5 ml tube. Post-‐ChIP analysis
53. Use 4 μl of eluted DNA for with QuantIT HS DNA Assay Kit (Invotrogen) 54. Run 5 μl of input DNA on a 2.2% agarose gel. 55. Use 3 μl DNA for qPCR.
Notes: 1) To optimize chromatin fragmentation, during sonication remove 10 μl aliquots of chromatin at various time points. Add 90 μl of IP Dilution Buffer, spin down for 10 min in microfuge, transfer supernatant to new tube and proceed with reverse X-‐linking, Proteinase K treatment and purify with Qiagen MinElute column as detailed for input in steps 35 to 50. Elute with 20 μl. Cell Lysis Buffer 5 mM HEPES pH 8.0 85 mM KCl 0.5% NP-‐40 (Pierce) Nuclei Lysis Buffer 50 mM Tris-‐Cl, pH 8.1 10 mM EDTA (pH 8.0) 1% SDS IP Dilution Buffer 0.01% SDS 1.1% Tween-‐20 (Pierce) 1.2 mM EDTA (pH 8.0) 16.7 mM Tris-‐Cl, pH 8.1 167 mM NaCl Chromatin Buffer 5 ml Nuclei Lysis Buffer 45 ml IP Dilution Buffer Low Salt Wash Buffer 0.1% SDS 1% Tween-‐20 2 mM EDTA 20 mM Hepes-‐KOH, pH 7.9 150 mM NaCl High Salt Wash Buffer 0.1% SDS 1% Tween-‐20 2 mM EDTA
20 mM Hepes-‐KOH, pH 7.9 500 mM NaCl LiCl Wash Buffer 100 mM Tris-‐HCl, pH 7.5 0.5 M LiCl 1% NP-‐40 1% Sodium Deoxycholate
