1. Thaw the nuclear pellet rapidly at 37 °C with gentle agitation.
2. Dilute the nuclear pellet to 2 mg/mL DNA with nucleus-isolation buffer and incubate the solution at 35 °C for 5 min with constant gentle agitation.
3. Add Micrococcal nuclease (25 U/mg of DNA) and digest for 10 min at 35 °C with constant gentle agitation to break up the chromatin in the nuclei.
4. Stop the digestion by adding 100 mM EGTA to a final concentration of 2.0 mM. Pellet the nuclei by centrifugation at 500 x g for 5 min at 4 °C and remove the supernatant.
5. To lyse the nuclei, resuspend the nuclear pellet in 10 mL of lysis buffer and mix vigorously on a vortex mixer.
6. Transfer the suspension of lysed nuclei to a dialysis bag and dialyze against 100 volumes of lysis buffer overnight at 4 °C.
7. Pellet the nuclear debris by centrifugation at 10,000 x g for 10 min at 4 °C and save the supernatant, which contains the solubilized chromatin (the chromatin includes from 1 to 10 nucleosomes). Measure the absorbance of a small aliquot in water at 260 nm to determine the concentration of chromatin DNA. The yield should be about 50% of the original total DNA (see Note 2). Add a cocktail of protease inhibitors to the solubilized chromatin.

Protocol A
8. Equilibrate hydroxyapatite in solution A and remove fine particles. Prepare a 20-mL hydroxyapatite column for FPLC. Equilibrate the column at 4 °C and wash it with 200 mL of 1% solution B (10 mM K-phosphate) at a flow rate of 8 mL/min using the FPLC system.
9. Apply no more than 15 mg (as DNA) of solubilized chromatin to the hydroxyapatite column at a flow rate of 4 mL/min in 1% buffer B. Wash the column with the same buffer for at least 20 min.
10. Elute material from the column under the following elution conditions: a linear gradient of 1% to 9% buffer B (10 to 90 mM K-phosphate) over 5 min; a linear gradient of 9% to 35% buffer B (90 to 350 mM K-phosphate) over 70 min (Hl-depleted chromatin is eluted); and a linear gradient of 35% to 100% buffer B (0.35 to 1.0 M K-phosphate) over 30 min (H1 is eluted), at a flow rate of 4 mL/min. The fraction collector should be programmed to collect 8-mL fractions.
11. Analyze the proteins in each fraction by SDS-polyacrylamide gel electrophoresis (18% polyacrylamide; see Note 3). Collect H1-depleted chromatin.
12. Dialyze the H1-depleted chromatin against TEP buffer overnight. Determine the concentration of DNA from the absorbance (OD_260nm = 20 corresponds to 1 mg/mL DNA). Concentrate the chromatin to 0.3 mg/mL (as DNA content) using a concentrator (MWCO, 30,000) for chromatin reconstitution, if necessary. Freeze the chromatin sample rapidly and store at －80 °C (see Note 4).

Protocol B
1. Dialyze the solubilized chromatin against K-phosphate buffer at 4 °C.
2. Add 4 mL of cation-exchange resin AG 50W-X2, equilibrated with K-phosphate buffer, to 20 mL of solubilized chromatin. Stir or shake gently at 4°C for 90 min.
3. Filter or decant the H1-depleted chromatin from the resin. Examine proteins in the preparation by SDS-PAGE (18% polyacrylamide; see Note 3).
4. Dialyze overnight against TEP buffer. Determine the concentration of H1-depleted chromatin DNA from the absorbance (OD_260nm =20 corresponds to 1 mg/mL DNA). Freeze the chromatin sample rapidly and store at －80 °C (see Note 4).