Yeast Chromatin Fractionation Protocol

1.     Grow 100mL of yeast to OD₆₀₀ ~1.0 in YPD (or appropriate selective media). 

2.     Harvest cells and wash in 50mL cold water.

3.     Resuspend cells in 3.0 ml 100 mM PIPES, pH 9.6, 10mM DTT and incubate at 30°C for 10 minutes.

4.     Spin down cells at 3000 rpm and resuspend in 2 ml spheroplasting buffer (0.6 M sorbitol, 50 mM KPO₄, pH 7.5, 10mM DTT) with 0.5 mM PMSF.

5.     Add 12 µl 100T zymolyase and shake at 30°C for 25 minutes.  If desired, monitor digestion by light microscopy or by reading A₆₀₀ of cells diluted in 1% SDS and allow to proceed until A₆₀₀ reaches ~10% of the starting material.

6.     Centrifuge at 3000 rpm for 5 minutes at 4°C.

7.      Gently resuspend pellets in 1 ml Diffley lysis buffer (20mM Pipes pH 6.8, 0.4 M sorbitol, 150 mM KOAc, 2 mM MgOAc) with 1.0 mM PMSF and other protease inhibitors.

8.     Centrifuge at 4000 rpm for 5 minutes, 4°C.

9.     Reusupend the pellet in 600 μl Diffley lysis buffer + 1% triton X-100.  Mix well and place on ice for 5 minutes.

10. Remove 200 μl to a new tube and store as “total” sample. Separate the remaining sample in to two tubes with 200 μl each. (**One is for analysis of the sup (soluble) and pellet (insoluble/chromatin) and one is for MNase digestion of pellet to verify chromatin association (see below).  If you don’t want to do this step, continue protocol with only one tube of 200 μl total.)

11. Spin the samples at 13K rpm for 15 minutes at 4°C.

12. Remove 100 μl of supernatant to a new tube and store as the soluble cell fraction.

13. Aspirate remaining supernatant from both tubes and wash the pellets in 100 μl Diffley lysis buffer + 1% triton X-100. (**Optional: Some chromatin-associated proteins may come off in this wash, so you can skip this step if your protein is loosely-associated with chromatin.)

14. Spin at 13K rpm for 10 minutes at 4°C.

15. Remove supernatant.  To analyze pellet (chromatin-associated fraction),  resuspend one pellet in 200 μl Diffley lysis buffer + 1% triton X-100.

To continue with MNase digestion to test release of protein from insoluble fraction….

16. Resuspend the other pellet in 200 μl Diffley lysis buffer + 1% triton X-100. Add CaCl₂ to 1mM final concentration.

17. Add MNase: 8 μl of 10U/mL stock.  Incubate at 37°C for 8 min.  (**The MNase concentration and time of incubation should be tested to see if the digestion releases mono-, di- and tri-nucleosomes.)

18. Stop the digestion by adding both EDTA to 1mM and EGTA to 1mM.

19. Centrifuge at 10K rpm for 5 minutes at 4°C.

20. Remove 75 μl of supernatant for Western blot (chromatin-associated proteins released following MNase-digestion) and remove 100 μl for PCI extraction and ethanol precipitation.  Check MNase-digestion of DNA on a 2% agarose gel.  

21. Aspirate remaining supernatant from pellet and reuspend in 200 μl Diffley lysis buffer + 1% triton X-100. 

22. For all samples, add 2X SDS loading buffer of the appropriate volume and boil for 5 minutes.  Load equivalent volumes on SDS-PAGE gels for Western analysis.  For most proteins, 10-20 μl of each sample is sufficient for Western analysis.