Reagents

100-bp DNA size ladder (Promega)

Agarose

Antisera (affinity-purified)

These should be raised against histone peptides with mono-, di-, or trimethylation or acetylation at a specific lysine/arginine residue of interest. In addition, include a control precipitation with an (IgG) antiserum that is not directed against chromatin proteins.

Cell culture from which nuclei are to be extracted (1 x 10⁷ to 1 x 10⁸ cells are required; see Steps 9-16)

Cell culture medium, appropriate for cells of interest (see Step 10)

ChIP elution buffer
ChIP incubation buffer
Dialysis-lysis buffer
DNA loading buffer (6X)

Ethanol (70%, v/v)
Ethidium bromide solution (20 mg/ml in H₂O)
Glycogen solution (20 mg/ml) (Roche)
Isopropanol
Liquid nitrogen (for frozen tissue only; see Step 1)
Micrococcal nuclease (MNase; 10 units/µl in 50% [v/v] glycerol) (Amersham Bioscience)

Store in 10- to 20-µl aliquots at –20°C. Each aliquot should be used only once to ensure equal enzyme activity in different chromatin preparations.

MNase digestion buffer
MNase stop solution

 NaCl (5 M)
Nuclei preparation buffer I, prechilled on ice
Nuclei preparation buffer II, prechilled on ice
Nuclei preparation buffer III, prechilled on ice
Phenol:chloroform:isoamyl alcohol 25:24:1 (v:v:v)

For extraction of genomic DNA, the phenol should be saturated beforehand with 100 mM Tris-Cl (pH 7.5) and stored at 4°C under 10 mM Tris-Cl (pH 7.5).

 Phosphate-buffered saline (PBS), cold

Protein A (e.g., CL-4B Sepharose from Amersham Bioscience) or protein G Sepharose

Protein A and protein G are bacterial cell wall proteins that bind to the Fc region of antibodies. These proteins are covalently coupled to Sepharose. The choice between protein A or protein G Sepharose depends on the nature of the antibody used for ChIP. In general, protein A works best for rabbit polyclonal antisera and for mouse monoclonal antibodies from the IgG2a, IgG2b, and IgG3 subclasses. Protein G Sepharose is preferred for mouse IgG1 monoclonal antibodies and for polyclonal antisera from mouse, rat, sheep, and goat.

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