Arabidopsis nuclei isolation and nuclear run-on (Gaudino and Pikaard, J. Biol. Chem. 272: 6799-6804; 1997)

1. Arabidopsis 2-week old plantlets (~800 plantlets) are harvested from 8 to 10 agar plates (standard germination medium without hormones), washed in ice cold sterile water, and kept at 4°C.

2. Plants are next submerged in cold diethyl ether for 5 minutes, then washed twice (5 min. each) in ice-cold sterile water.

3. Plants are homogenized in 100 ml (approximately 3 volumes) of nuclei isolation buffer (1 M sucrose, 50 mM Tris-HCl [pH 7.2], 5 mM MgCl2, 5 mM KCl2, 10 mM 2-mercaptoethanol, 0.2 mM PMSF) with a motorized homogenizer (Fisher Scientific Powergen 700).

4. The homogenate is filtered through 8 layers of cheesecloth, 1 layer Miracloth (Calbiochem), and 1 layer of 50 um nylon mesh.

5. The filtrate is centrifuged at 14, 000 x g for 15 minutes at 4°C.

6. The pellet is resuspended in 10 ml of nuclei isolation buffer using a 15 ml. Dounce homogenizer (A pestle), and the final volume is measured. One volume of "100%" Percoll solution ( 34.23 g sucrose, 5 ml 1 M Tris-HCl [pH7.2], 0.5 ml 1 M MgCl2, 0.5 ml 1 M KCl, 34 ul 2-mercaptoethanol, 100 ul 2 mM PMSF, and Percoll to 100 ml) is added to 19 volumes of nuclei.

7. 7 ml of crude nuclei in 5% Percoll solution is then layered onto a 4-step discontinuous Percoll gradient with 5 ml layers each of 15%, 30%, 45%, and 60% Percoll solutions (diluted from the 100% Percoll solution using nuclei isolation buffer).

8. Gradients are centrifuged at 2000 rpm for 10 minutes, then 8000 rpm for 20 minutes in a Beckman SW 28 rotor at 4°C. Nuclei are harvested from the 30%/45% and 45%/60% boundaries and pooled (both are transcriptionally equivalent).

9. Nuclei are diluted with 5 volumes of nuclei isolation buffer, mixed by inversion, and collected by centrifugation at 1500x g, 10 minutes, 4°C in a clinical tabletop centrifuge (Beckman).

10. Nuclei are resuspended gently in 50 ml (approximately 25 volumes) of nuclei isolation buffer, and again collected by centrifugation. Final resuspension is in 1 ml of nuclei storage buffer (50 mM HEPES [pH 7.2], 5 mM MgCl2, 5 mM KCl, 2 mM DTT, 0.2 mM PMSF, 50% glycerol).

11. Aliquots are transferred to pre-chilled 1.5 ml microcentrifuge tubes, frozen in liquid nitrogen, and stored at -80°C.

Nuclear run-on.

1. Frozen nuclei are thawed on ice, stained with 4', 6-diamidino-2-phenylindole (DAPI), and counted in a hemacytometer using fluorescence microscopy. Nuclear run-ons for filter hybridizations involve one million nuclei; for total incorporation experiments, 100,000 nuclei are used.

2. Nuclear run-on reactions are for 10 minutes at 30°C in a final volume of 50 ul (40 ul of run-on reaction buffer, 10 ul nuclei). Nuclear run-on reaction buffer is: 50 mM Tris-HCl [pH 7.2], 5 mM MgCl2, 5 mM KCl, 75 mM (NH4)2SO4, 1 mM MnCl2, 300 uM each ATP, GTP, UTP, 4.5 uM CTP, 0.5 uM a-32P CTP, 5 mM DTT, 0.2 mM PMSF, 2 units RNasin RNase inhibitor (Promega), 10% glycerol. (If pol II and pol III are to be inhibited, 150 ug/ ml alpha-amanitin is used (increasing the amanitin level to 500 ug/ml was found to have no additional effect).

3. 5 units of RNase-free DNase (Promega RQ1 DNase) are then added and the incubation is continued 10 minutes.

4. 250 ul of 2 mg/ml pronase, 1% SDS is added and reactions are incubated for 30 minutes at 55°C.

5. Reactions are extracted twice with phenol/chloroform, and once with chloroform.

6. Ammonium acetate is added to a final concentration of 2 M and RNA is precipitated with 2.5 volumes of ice cold 100% ethanol.

7. Following centrifugation, RNA is pelleted and washed twice with 70% ethanol.

8. Dried pellets are resuspended in filter hybridization solution, formamide RNA loading buffer, or TE (pH 7.2) for hybridization, polyacrylamide gel electrophoresis, or scintillation counting, respectively.

References
Feinbaum, R. L., and Ausubel, F. M. (1988) Mol. Cell. Biol. 8, 1985-1992
Guilfoyle, T. J. (1995) Methods Cell. Biol. 50, 101-112