Nuclei Preparation from Tissues and Cells

Steps 1-8 describe the purification of nuclei from tissue, while Steps 9-16 describe the purification of nuclei from cultured cells. To prevent chromatin degradation, all steps of the nuclei purification procedure should be performed on ice, or at 4°C (e.g., precool the centrifuge rotors). In addition, one set of micropipettes should be dedicated only to the preparation of nuclei, chromatin, and ChIP analysis, to avoid DNA contamination. Wear gloves throughout all procedures, and respect the safety rules, especially when handling phenol.

Purification of Nuclei from Tissue (2 h)

1. Dissect fresh tissue (maximum 0.2 g in total) and rinse it in cold PBS.
See Troubleshooting. For many tissue types, frozen tissue (snap-frozen in liquid nitrogen) can be used as well. This tissue should first be crunched into powder in a mortar filled with liquid nitrogen; this powder is used for Step 2. The mortar should be prechilled with liquid nitrogen and the tissue kept constantly under liquid nitrogen.

2. Homogenize the tissue in a prechilled glass homogenizer with 5-10 ml of ice-cold nuclei preparation buffer I, until no clumps of cells persist (~10-20 strokes). Filter the suspension through four layers of muslin cheesecloth moistened beforehand with nuclei preparation buffer I.

3. Transfer the cell suspension to a 14-ml polypropylene tube, and centrifuge the samples in a swing-out rotor (3000g, 5 min, 4°C).

4. Pour off the supernatant, and resuspend the cells in 2 ml of ice-cold nuclei preparation buffer I. Add 2 ml of ice-cold nuclei preparation buffer II, mix gently, and place the tubes on ice a maximum of 5 minutes.
See Troubleshooting.

5. Prepare two new 14-ml polypropylene tubes, each containing 8 ml of ice-cold nuclei preparation buffer III. Carefully layer 2 ml of each cell suspension (from Step 4) onto each 8-ml sucrose cushion. Cover each tube with a piece of Parafilm.

6. Centrifuge the tubes in a prechilled swing-out rotor (10,000g, 20 min, 4°C).
The nuclei will form a pellet at the bottom of the tube, whereas the cytoplasmic components will remain in the top layer. At this step, the nuclear pellet should be white.

7. Carefully take off the supernatant with a Pasteur pipette.
This is a critical step, as the top solution (which contains the detergent IGEPAL CA-630) should not come into contact with the nuclear pellet at the bottom of the tube. One way to achieve this is to remove the supernatant in about three steps, changing the Pasteur pipette each time.
See Troubleshooting.

8. Resuspend the nuclear pellet in 1 ml of MNase digestion buffer, and keep the samples on ice. If possible, MNase digestion (Step 23) should be started immediately. Nuclei can be stored for up to 1 day at 4°C.
At this point, the nuclei can be counted using a microscope slide for counting cells. The number of nuclei obtained per gram of tissue varies according to tissue type. For liver, for example, this protocol yields ~2 x 10⁹ nuclei/g tissue.

Nuclei Preparation from Cultured Cells (2 h)

9. Culture 1 x 10⁷ to 1 x 10⁸ cells. Ensure that the cells are not grown beyond semiconfluency.

10. Rinse the cells in PBS, add 2 ml of trypsin solution (for adhering cells only), and incubate them at 37°C. When trypsination is complete, stop the reaction by adding 5 ml of culture medium to the cells.

11. Divide the cell suspension between two 14-ml polypropylene tubes, and centrifuge the samples in a swing-out rotor (4000g, 5 min, 4°C).

12. Pour off the supernatant, and resuspend the cells in 2 ml of ice-cold nuclei preparation buffer I. Add 2 ml of ice-cold nuclei preparation buffer II, mix gently, and place the tubes on ice a maximum of 5 minutes.
See Troubleshooting.

13. Prepare two new 14-ml polypropylene tubes, each containing 8 ml of ice-cold nuclei preparation buffer III. Carefully layer 2 ml of each cell suspension (from Step 4) onto each 8-ml sucrose cushion. Cover each tube with a piece of Parafilm.

14. Centrifuge the tubes in a prechilled swing-out rotor (10,000g, 20 min, 4°C).
The nuclei will form a pellet at the bottom of the tube, whereas the cytoplasmic components will remain in the top layer. At this step, the nuclear pellet should be white.

15. Carefully take off the supernatant with a Pasteur pipette.
This is a critical step, as the top solution (which contains the detergent IGEPAL CA-630) should not come into contact with the nuclear pellet at the bottom of the tube. One way to achieve this is to remove the supernatant in about three steps, changing the Pasteur pipette each time.
See Troubleshooting.

16. Resuspend the nuclear pellet in 1 ml of MNase digestion buffer, and keep the samples on ice. If possible, MNase digestion (Step 23) should be started immediately. Nuclei can be stored for up to 1 day at 4°C.
At this point, the nuclei can be counted using a microscope slide for counting cells. The number of nuclei obtained per gram of tissue varies according to cell type.

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