Proteinase K (10 mg/ml) (optional; see Step 70)

Sodium butyrate (optional; for analyzing histone acetylation only)

To analyze histone acetylation, we recommend adding sodium butyrate (to a final concentration of 5 mM) to the solutions used for the purification of nuclei and for the preparation of input chromatin. Sodium butyrate prevents loss of histone acetylation via the nonspecific action of endogenous histone deacetylases.

 Sodium dodecyl sulfate (SDS; 10 %, w/v)

TBE buffer (1X)
TE buffer (1X, pH 7.5)
Tissue samples (fresh or frozen) from which nuclei are to be extracted (see Steps 1-8)
Trypsin solution (0.05% [w/v]) (Sigma)

 Tubing preparation solution I
Tubing preparation solution II

Washing buffer A
Washing buffer B
Washing buffer C

Equipment

Centrifuges:

Bench-top centrifuge with cooling system for 1.5-ml microcentrifuge tubes
Centrifuge with a swing-out bucket rotor for 15-ml polypropylene tubes
High-speed centrifuge with cooling system and a swing-out bucket rotor for 14-ml polypropylene tubes

Dialysis tubing (0.5-mm thick, 10-kDa pore width) (VWR international)
Homogenizer, prechilled on ice

We use a tissue grinder/homogenizer (from BDH) that has a glass mortar (tube) and a pestle with a hard plastic head. The clearance between pestle and mortar is 0.15-0.25 mm.

Horizontal gel electrophoresis tank for agarose gels

Ice
Magnetic stirrer (see Step 32)
Microcentrifuge tubes (1.5 ml and 2.0 ml)

Chromatin immunoprecipitations and incubations with Protein A (G) Sepharose (Steps 44-69) are performed in microcentrifuge tubes. These tubes may be siliconized beforehand (e.g., with a 2% [v/v] dichloromethylsilane solution) in order to prevent nonspecific association of chromatin and antibodies to the inner walls of the tubes. In our laboratory, we have obtained comparable results with nonsiliconized and siliconized microcentrifuge tubes.

Microscope, inverted light (optional; see Step 8)
Mortar and pestle, prechilled in liquid nitrogen (for frozen tissue only; see Step 1)
Muslin cheesecloth

Prepare the cheesecloth by rinsing with H₂O and then autoclaving.

Parafilm
Pasteur pipettes
Polypropylene tubes, 14 ml (e.g., 17 x 100-mm Falcon tubes) and 15 ml (e.g., 17 x 120-mm Falcon conical tubes)
Rotating wheels at 4°C and room temperature
Spectrophotometer
Tray for staining gels (see Step 41)
Universal tubing clamps (5 mm) (Spectrum Laboratories)
UV lamp
Vortex mixer
Water bath set at 37°C

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