1. Harvest HeLa S3 cells from a culture of approximately 5 × 10⁵ cells/mL in a 2-L spinner flask by centrifugation at 500 x g for 10 min at 4 °C.
2. Wash the cell pellet by gentle resuspension in ice-cold PBS and repeat the centrifugation as above.
3. Wash the cell pellet again with ice-cold nucleus-isolation buffer and resuspend the cell pellet by gentle agitation in 50 mL of ice-cold nucleus-isolation buffer supplemented with 0.5% (v/v) Triton X-100. Allow the cells to swell for 10 min on ice.
4. Homogenize the swollen cells on ice with 15 strokes of a Dounce homogenizer on ice. Pour the suspension of homogenized cells (about 60 mL) into two 50-mL conical tubes and centrifuge them at 500 x g for 5 min at 4 °C.
5. Pour off cloudy supernatants slowly and then gently resuspend each nuclear pellet in approximately 5 mL of nucleus-isolation buffer plus Triton X-100. Add nucleus-isolation buffer to bring the suspension to the original volume (approximately 30 mL per tube) and pellet the nuclei again by centrifugation at 500 x g for 5 min at 4 °C.
6. Repeat step 5, at least twice, until the nuclei are pure white and the supernatant is clear.
7. Resuspend each nuclear pellet gently in approximately 5 mL of nucleus-isolation buffer and combine the suspensions in one conical tube. Adjust the total volume to approximately 20 mL with nucleus-isolation buffer. Remove a 5-μL aliquot and measure the absorbance at 260 nm in 1 mL of a saturated solution of NaCl and urea, using this solution as a blank, to quantify the amount of chromatin DNA (20 OD_260nm = 1 mg/mL DNA). The total yield should be close to 30 mg of chromatin as DNA. Pellet the nuclei by centrifugation at 10,000 x g and carefully remove the supernatant. Freeze the nuclear pellet in liquid nitrogen and store at －80 ° C.