Yeast Nuclei Isolation

Adapted from Hahn Lab Protocol

Grow 2-4 Liters of yeast in appropriate medium to OD600 ~2-5 (2.5 works nicely). Works best if you innoculate cultures the night before so that they are at appropriate OD the next morning (40 ml of dense RMY200 culture into 2L YPD will give OD600 = 2.5 after ~16 hours).

Harvest cells in 500 ml bottles (don't fill more than 3/4 of the way up or they will leak) at 4000 RPM for 5 minutes in JA-10 rotor.

Resuspend in 35ml Zymolyase Buffer (leave in 500 ml bottle), and add 1 ml 1M DTT (final concentration is 3 mM)

Incubate at 30oC for 15 minutes with gentle shaking (~100 RPM on shaker works well)

Pellet cells at 4000 RPM for 5 minutes in JA-10.

Resuspend in 30 ml Zymolyase Buffer, and add 20 ml YPD/S.

Add 25mg Zymolyase 100T and incubate at 30oC for 1.5 hours with gentle shaking. (Test for completion of spheroplasting by mixing 4 ul of spheroplasts with 4 ul 1% SDS on a slide. Cells are done spheroplasting if more than 80% are lysed and form ghosts.)

Add 100 ml YPD/S and pellet cells at 4000 RPM for 10 minutes.

Resuspend cells in 250 ml YPD/S (room temp). (Works best if you add 50 ml and use a weighing spatula to dislodge cells and then add rest of YPD/S. Spheroplasts are sticky and clump together, however the pellet is loose and care should be taken not to lose it. Pipetting up and down may disrupt cells.)

Pellet cells at 4000 RPM for 5 minutes in cold.

Resuspend in 250 ml YPD/S (ice cold).

Pellet cells at 4000 RPM for 5 minutes in cold. (Cells may be left on ice for about an hour at this point, if needed.)

Resuspend in 200 ml 1M Sorbitol (ice cold).

Pellet cells at 4000 RPM for 5 minutes in cold.

Resuspend in 100 ml Buffer N (ice cold). (If you will not be using the nuclei the same day and would like to freeze them, make sure and put aside 5 ml of cold Buffer N for resuspending nuclei.)

Wash Yamamoto Homogenizer (in cold room) by passing 500 ml cold Hall Water through it at 1000 RPM. Pass cells through homogenizer at 1000 RPM five times. Wash homogenizer thoroughly with 500-1000 ml cold Hall Water.

Transfer to 30 ml JA-20 tubes (works best if you only fill 2/3 per tube).

Pellet cellular debris at 2000 RPM for 15 minutes at 4oC in JA-20.

Transfer supernatant to new tubes. (Try to avoid transferring slimy white gunk at bottom of tube. If you want cleaner prep you can repeat 2000 RPM spin.)

Pellet nuclei by spinning at 6000 RPM for 25 minutes at 4oC in JA-20.

Discard supernatant.

Resuspend nuclei in 1 ml Buffer N (or appropriate buffer) by pipetting up and down. Nuclei may now be frozen in liquid nitrogen and stored at -80oC.

To measure yield, add 10 ul nuclei to 990 ul 2M NaCl/5M Urea and measure A260. You can check quality of nuclei under light microscope or with DAPI by adding 4 ul nuclei to 4 ul 1ug/ml DAPI and viewing under mercury lamp.

Buffers:

Zymolyase Buffer:

1M Sorbitol
50mM Tris-HCL pH 7.5
10mM MgCl2

YPD/S:

YPD
1M Sorbitol

Buffer N:

25mM K2SO4
30mM HEPES pH 7.6
5mM MgSO4
1mM EDTA
10% Glycerol
0.5% NP40
7.2mM Spermidine Hexahydrate (phosphate salt)
3mM DTT
1x Protease Inhibitors Cocktail