JJG Lab ChIP-Seq protocol by Martiniano Ricardi
Half-native nuclei prep
- 1. Fix 2.5-5 g of grinded fruit tissues with 50 ml ice-cold Sucrose Buffer with 1% formaldehyde (ripened fruit has less DNA, e.g. 0.5-1 g is ok for 7DPA fruit, but Br+10 fruit might need 10 g, formaldehyde can be added afterward)
- 2. Mixed well and incubate on ice for 1min
- 3. Add Glycine to 0.125 M (dissolve the appropriate amount of glycine in a small volume of buffer first), mix well, filter through the 110 um Nylon mesh
- 4. Spin 2000 rpm for 15 min at 4C
- 5. Pour off the supernatant and keep the nuclei pellet
- 6. Resuspend in 20 ml ice-cold Sucrose Buffer, transfer to 2 ml tubes (10 in total)
- 7. Spin 5,000 rpm for 10 min at 4C
- 8. Resuspend the nuclei in 4 ml of Percoll 95% buffer.
- 9. Spin 12,000xG for 10 min at 4C
- 10. Take the upper phase into a falcon tube (all the solids you may find there) without disturbing the bottom pellet (if there are diffuse solids along the whole tube more percoll buffer is needed). You can use a spatula if the upper phase is to solid.
- 11. Dilute to 1/4 with Nuclei Wash buffer and place in as many 2ml tubes as needed
- 12. Spin 12,000xG for 10 min at 4C
- 13. Remove all the supernatant and keep the pellet
- 14. Resuspend the pellets all together with 2-4 mL of the MN buffer and split into tubes with 1ml volume.
- 15. Add 50 uL of RNase A to each 1 mL of the nuclei, mix well, incubate at 37C water bath for 10 min
- 16. Add 2 uL Micrococal Nuclease (NEB), mix well, incubate at 37C water bath for 10-20 min
- 17. Add EDTA to 10 mM to stop the reaction, immediately placed on ice.
- 18. Sonicate the nuclei using probe-base sonicator (5 pluse, 10% output) to disrupt the nucleus membrane (keep all the tube on ice all the time)
- 19. Add NaCl to 500 mM, SDS to 0.1% and TritonX-100 to 1%, rotate at 4C for 30 min to release the mononucleosome.
- 20. Spin 15 min 4oC 14,000 rpm to get rid of the debris and gel-like pellet, repeat once more
- 21. Add 2 Volumes of TE to bring down the NaCl, SDS and TritonX concentration
- 22. Spin 15 min 4C 14,000 rpm again to remove the debris
- 23. Keep 5% nuclei as input (for reverse-crosslinking and check DNA concentration)
- 24. Use 1,8 ml of chromatin per ChIP assay.
Prepare antibody and Dynabead A (or protein G for monoclonal Ab)
- 1. Wash 50 µl beads 3 times with chilled 200 uL wash buffer.
- 2. Add 2-4 uL (2 for monoclonal and 4 for polyclonal) of the Ab to 30 uL of the beads, top up with wash buffer to 200 uL.
- 3. Add the remaining 20 uL beads to pre-clear the DNA.
- 4. Rotate the Ab and chromatin tubes at 4C for 2 h.
- 5. Wash the Ab-beads twice with 200 uL of Washing Buffer, mix well and incubate 5-10 min between wash.
- 6. Resuspend the Ab-beads in 50 uL of Washing Buffer and keep on ice.
- 7. Put the chromatin tube with beads onto the magnetic-stand, transfer the supernatant to a new 2 mL tube. (spin them if there are still jelly-like debris)
- 8. Add the 50 uL Ab-beads to the chromatin solution, add BSA to 100ug/ml, DNA oligos to 5 ng/uL and extra Protease inhibitor (to 1x final concentration) and rotate O/N at 4C.
Wash and reverse-crosslinking
- 1. Wash 4 times with 1 mL of washing buffer, pool identical chip-samples into a single 2 ml tube, incubate 10 min and change tube between wash.
- 2. Wash with TE once.
- 3. Add 100 uL elution buffer, transfer to PCR tube.
- 4. Heat 55C 1 h, 65 oC 4h.
- 5. Collect the solution on a magnet, wash the beads again with 200 uL elution buffer.
- 6. Combine the 2 elute and extracted with Phenol-Chloroform once.
- 7. Precipitate with 0,1 volumes of NaAc, 1 uL glycogen, and 3 volumes of Ethanol.
- 8. Dry well the pellet and resuspend with 20 uL of TE.
- 9. Add 1.8 volumes of AMPure XP (before pippeting make resuspend them by vortexing).
- 10. Mix the samples well up and down with the pipette.
- 11. Leave 15’ to room temperature.
- 12. Collect with magnetic rack and take all the liquid.
- 13. Wash twice with 200ul of EtOH 75% keeping the tube always on the magnetic rack. Do not disturb the pellet during the washes.
- 14. Dry well but not too much.
- 15. Elute with 20 uL H2O. Use 1-2 uL (dilute to 20 uL) for Q-PCR, store the rest for Illumina sequencing library construction in LOW-BIND tube.
Sucrose Extraction Buffer 1
| 0.4 M sucrose | 13.69 gr / 100 ml |
| 10 mM Tris-HCl pH 7.5 | 1/100 dil from 1M stock |
| 10 mM MgCl2 | 1/100 dil from 1M stock |
| Components that should be added fresh: |
| 5 mM BME | 1/2854 dil from 14.27 M stock |
| 1% Triton X-100 (can’t be autoclaved) | % Triton X-100 (can’t be autoclaved) |
| 1 mM PMSF | 1/200 dil from 0,2M stock |
| 0.25 x Proteinase inhibitor | 1/400 dil from 100x stock or use a tablet |
Nuclei Wash buffer
| 10% Glycerol | 1/10 dil from 100% Stock |
| 50 mM Tris-Cl pH 7.5 | 1/20 dil from 1M Stock |
| 1 mM CaCl2 | 1 mM CaCl2 |
| Components that should be added fresh: |
| 0.1% Triton X-100 | 1/100 dil from 10% stock |
| 0.5 mM PMSF | 1/400 dil from 0.2M stock |
| 1/400 dil from 0.2M stock | 1/100 dil from 100x stock |
MN digestion buffer
| 50 mM Tris-Cl pH 7.5 | 1/20 dil from 1M stock |
| 1 mM CaCl2 | 1/100 dil from 0.1M Stock |
| Components that should be added fresh: |
| 0.1% Triton X-100 | 1/100 dil from 10% stock |
| 0.5 mM PMSF | 1/400 dil from 0.2M stock |
| 1 x Proteinase inhibitor | 1/100 dil from 100x stock |
Wash Buffer
| 10 mM Tris-HCl pH 7.5 | 1/100 dil from 1M stock |
| 150 mM NaCl | 1/33.33 dil from 5M stock |
| 1 mM EDTA | 1/500 dil from 0.5M stock |
| Components that should be added fresh: |
| 1 x BSA | 1/100 dil |
| 5 ng/uL DNA oligo | 1/100 dil for 100uM stock (20nt oligos) |
| 1% Triton X-100 | 1/100 from 100% stock |
| 1 x Proteinase inhibitor | 1/100 from 100x stock |
Elution Buffer
| 250 mM NaCl | 1/20 dil from 5M stock |
| 100 mM Tris-Cl pH 7.5 | 1/10 dil from 1M stock |
| 10 mM EDTA | 1/50 dil from 0.5 stock |
| 0.2% SDS | 1/100 dil from 20% stock |
| 10ngr/ul Proteinase K | 1/1000 dil from 10 ugr/ul stock |
TE Buffer ph 7.5
| 100 mM Tris-Cl pH 7.5 | 1/10 dil from 1M stock |
| 10 mM EDTA | 1/50 dil from 0.5 stock |
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