Nucleosome ChIP Protocol
- 7 steps
- 1 hours
- 7 reagents required
This method allows ChIP analysis at nucleosome resolution by taking advantage of the fact that micrococcal nuclease (MNase) can efficiently digest cross-linked chromatin. It is applicable to study histone modifications and factor occupancy on nucleosomes, but not if the factors are recruited to naked DNA sequence. However, it should be noted that if two neighboring nucleosomes are bridged via intermediary factors, they are not expected to be resolved. To investigate individual nucleosomes, the sonication step of the standard ChIP protocol is replaced by micrococcal nuclease digestion of cross-linked chromatin. Complete digestion (i.e. to obtain mononucleosome-sized fragments) requires pure nuclei. While nuclei obtained by the standard ChIP protocol step 7 is often fine, we obtain more reproducible results by including the steps below.
Reagents Required
| Product | Preparation |
| Sucrose Buffer A | 0.32 mM sucrose 15 mM Hepes, pH 7.9 60 mM KCl 2 mM EDTA 0.5 mM EGTA 0.5% BSA 0.5 mM spermidine 0.15 mM spermine 0.5 mM DTT |
|
Sucrose Buffer B Note: This is the same as Sucrose Buffer A without BSA |
0.32 mM sucrose 15 mM Hepes, pH 7.9 60 mM KCl 2 mM EDTA 0.5 mM EGTA 0.5 mM spermidine 0.15 mM spermine 0.5 mM DTT |
| Buffer NUC | 15 mM Hepes, pH 7.5 60 mM KCl 15 mM NaCl 0.34 mM sucrose 0.15 mM mercaptoethanol 0.15 mM spermine 0.5 mM spermidine |
| NaOH | Prepare 1M NaOH. |
| CaCl2 | |
| Micrococcal nuclease (MNase) | |
| 2X Sonication Buffer X | 60 mM Hepes, pH 7.9 220 mM NaCl 10 mM EDTA 2% Triton X-100 0.2% Na-deoxycholate 0.2% SDS 0.5 mM PMSF Protease inhibitor cocktail (Roche) |
Procedure
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After step 6 of the standard ChIP protocol, resuspend cells in at least 10 pellet volumes of Sucrose Buffer A and perform dounce homogenization (10 up-downs). Check nuclei by trypan blue staining.
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Layer the nuclear suspension over an equal volume of Sucrose Buffer B and centrifuge for 15 minutes at 3,000 rpm. This is performed in 15 mL tubes, 5 mL nuclear suspension + 5 mL Sucrose Buffer B.
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Remove the supernatant stepwise with a 1 mL pipette and collect purified nuclei from the bottom of the gradient by resuspension in 1 mL Buffer NUC. Transfer to a clean tube.
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Wash nuclei with buffer NUC once and resuspend them in Buffer NUC to obtain 0.2 OD260 per 5 µL sample. OD is measured by diluting 5 µL nuclei in 1 mL 1M NaOH.
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Add CaCl2 to 3 mM final concentration and immediately add 100 units/mL MNase. In most cases, this amount of MNase is sufficient to obtain mononucleosome-sized fragments but may need to be optimized for the particular experiment.
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Incubate for 5 minutes at 37°C and stop reactions by the addition of an equal volume of 2X Sonication Buffer X.
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Vortex and check lysis in a microscope. If not completely lysed, perform one short (5 second) sonication. Centrifuge at 14000 rpm for 15 minutes. Take the soluble chromatin-containing supernatant and proceed to step 14 of the standard ChIP protocol.
Note: At the end of the procedure the purified DNA fragments are around 146 bp in size. To design primers for PCR analysis, the positions of the nucleosomes should be determined. In our experience, low-resolution mapping by indirect end-labeling of partially digested chromatin with MNase is sufficient.