MNase Digestion for Nucleosome Mapping in Neurospora

Materials and Reagents

1. Filter paper (VWR International, catalog number: 1001-090 )
   
2. D-(+)-glucose monohydrate (Sigma-Aldrich, catalog number: 49159 )
   
3. L-arginine-¹³C₆,¹⁵N₄ hydrochloride (Sigma-Aldrich, catalog number: 608033 )
   
4. 1x Vogel’s salt (Vogel, 1956; Vogel, 1964)
   
5. D-biotin (Thermo Fisher Scientific, Molecular Probes™, catalog number: B1595 )
   
6. Sucrose (Sigma-Aldrich, catalog number: S0389 )
   
7. KCl (Thermo Fisher Scientific, Ambion™, catalog number: AM9640G )
   
8. NaCl (Thermo Fisher Scientific, Invitrogen™, catalog number: 24740011 )
   
9. Tris hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: 10812846001 )
   
10. MgCl₂ (Thermo Fisher Scientific, Ambion™, catalog number: AM9530G )
    
11. Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: 793639-500G )
    
12. NP-40 (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 85124 )
    
13. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo Fisher^TM, catalog number: 20290 )
    
14. Spermidine (Sigma-Aldrich, catalog number: S0266 )
    
15. HEPES (Sigma-Aldrich, catalog number: H4034 )
    
16. EGTA (Sigma-Aldrich, catalog number: E3889 )
    
17. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
    
18. EDTA (Thermo Fisher Scientific, Thermo Fisher^TM, catalog number: 17892 )
    
19. Glycerol (Sigma-Aldrich, catalog number: G6279 )
    
20. Glycogen, molecular biology grade (Thermo Fisher Scientific, Thermo Fisher^TM, catalog number: R0561 )
    
21. Ethanol
    
22. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    
23. Glycine (Thermo Fisher Scientific, Invitrogen™, catalog number: 15527013 )
    
24. Nuclease free H₂O (Thermo Fisher Scientific, Thermo Fisher^TM, catalog number: R0581 )
    
25. Liquid nitrogen
    
26. Agarose (Thermo Fisher Scientific, Fisher Scientific^TM, catalog number: BP1356100 )
    
27. MNase (Sigma-Aldrich, catalog number: N3755-500 )
    
28. RNAse cocktail enzyme mix (Thermo Fisher Scientific, Invitrogen™, catalog number: AM2286 )
    
29. Proteinase K solution (Thermo Fisher Scientific, Invitrogen™, catalog number: AM2548 )
    
30. Wizard^® SV gel and PCR clean-up system (Promega Corporation, catalog number: A9281 )
    
31. EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 0 5892791001 )
    
32. C₆H₈O₇
    
33. ZnSO₄^.7H₂O
    
34. Fe(NH₄)₂(SO₄)₂^.6H₂O
    
35. CuSO₄^.5H₂O
    
36. MnSO₄^.1H₂O
    
37. H₃BO₃ (anhydrous)
    
38. Na₂MoO₄^.2H₂O
    
39. Chloroform
    
40. MilliQ water
    
41. Na₃C₆H₅O₇
    
42. KH₂PO₄
    
43. NH₄NO₃
    
44. MgSO₄^.7H₂O
    
45. CaCl₂^.2H₂O
    
46. Liquid medium (see Recipes)
    
47. Trace element solution (see Recipes)
    
48. 50x Vogel’s salt (see Recipes)
    
49. Lysis buffer (see Recipes)
    
50. MNase resuspension buffer (see Recipes)
    
51. Stop buffer (see Recipes)
    
52. TE buffer (see Recipes)
    

Equipment

1. Centrifuge
   
2. Vortex
   
3. Mortar and pestle
   
4. Thermo-mixer (Eppendorf AG, model: Thermo-mixer R )
   
5. Buhner funnel (Thermo Fisher Scientific, Fisher Scientific^TM, catalog number: FB966B )
   
6. Shaking incubator
   
7. Agarose gel electrophoresis
   

Procedure

1. Inoculate ~ 2 x 10⁸ conidia of Neurospora crassa to 200 ml liquid medium in a 500 ml flask.
   
2. Grow the culture with 100 rpm shaking under desired light and temperature conditions. Representative cultures were grown for three days at 24 °C and at 80 µE light intensity.
   
3. In order to crosslink, add paraformaldehyde to the culture to a final concentration of 1% and incubate for 10 min shaking at 24 °C.
   
4. Quench paraformaldehyde for 5 min with glycine at final concentration of 125 mM.
   
5. Filter liquid medium through Buhner funnel and rinse mycelia with 500 ml of water (room temperature). Squeeze mycelia between tissue paper to get rid of all water and then place the mycelia in Eppendorf tube and immediately freeze in liquid nitrogen. At this step mycelia can be stored at -80 °C.
   
6. Cool mortar and pestle by submerging in liquid nitrogen for 5 min. Take the mortar and pestle out and then place frozen mycelia immediately into mortar and grind mycelia with pestle until it is finely ground powder. At this step one should avoid to warm mycelia/ground mycelia powder.
   
7. Resuspend ~400 mg ground mycelia in 3.75 ml of lysis buffer with freshly added 0.5 mM DTT, 0.5 mM spermidine and EDTA-free protease inhibitor tablets.
   
8. Mix the suspension well by vortexing and incubate on ice for 5 min.
   
9. Distribute the suspension into five 1.5 ml Eppendorf tubes (750 μl per tube).
   
10. Resuspend the MNase powder in 850 μl of MNase resuspension buffer (estimated concentration=0.58 U/μl). Aliquots can be stored at -20 °C. Avoid freeze-and-thaw cycles.
    
11. Add respectively 0 [control], 0.75, 1.5, 3, 6 Units of MNase to each tubes. Incubate all samples at 25 °C for 1 h while shaking at 400 rpm in a Thermo-mixer.
    
12. Add 85 μl of stop buffer to each sample to stop digestion and centrifuge at 20,000 x g for 20 min to pellet the cell debris. Transfer supernatants to new tubes
    
13. Add 2 μl of RNase cocktail enzyme mix to each supernatant and incubate at 37 °C for 45 min shaking at 400 rpm in a Thermo-mixer.
    
14. Add 15 μl of Proteinase K solution to each sample and incubate at 65 °C for 2 h shaking at 400 rpm in a Thermo-mixer.
    
15. Add 40 μg glycogen and 1,250 μl of 100 % EtOH to each sample and incubate at -20 °C for 30 min.
    
16. Perform centrifugation at max speed (~20,000 x g) for 30 min at 4 °C. Discard supernatant. Wash the DNA pellet with 700 μl 70 % ice-cold ethanol. Remove the ethanol slowly without disturbing the pellet. Drying the pellet is not required.
    
17. Dissolve the washed DNA pellet in 100 μl DNAse/RNAse free H₂O.
    
18. Further purify DNA samples by using PCR-clean up kit.
    
19. Load samples on 1.7% agarose gel (80 volts for 1 h) to analyze nucleosomal ladder (Figure1).
    

Representative data

Figure 1. Representative image of MNase digested and purified Neurospora nucleosomal DNA loaded on 1.7% agarose gel

Recipes

1. Liquid medium (prepare freshly)
   2% (w/v) glucose monohydrate
   0.5 % (w/v) L-arginine
   1x Vogel’s salt (Vogel, 1956; Vogel, 1964)
   10 ng/ml biotin
   
2. Trace element solution (stored at RT)
   Dissolve indicated amount in 95 ml MilliQ water
   5 g C₆H₈O₇
   5 g ZnSO₄^.7H₂O
   1 g Fe(NH₄)₂(SO₄)₂^.6H₂O
   0.25 g CuSO₄^.5H₂O
   0.05 g MnSO₄^.1H₂O
   0.05 g H₃BO₃ (anhydrous)
   0.05 g Na₂MoO₄^.2H₂O
   Add 1 ml chloroform as preservative
   
3. 1 L 50x Vogel’s salt (stored at RT)
   Dissolve indicated amount in 755 ml MilliQ water
   125 g Na₃C₆H₅O₇
   250 g KH₂PO₄
   100 g NH₄NO₃
   10 g MgSO₄^.7H₂O
   5 g CaCl₂^.2H₂O
   5 ml of trace element solution
   250 µg biotin
   No pH adjustment is necessary
   Add 5 ml chloroform as preservative
   
4. Lysis buffer (stored at 4 °C)
   250 mM sucrose
   60 mM KCl
   15 mM NaCl
   15 mM Tris-HCl (pH 7.4)
   3 mM MgCl₂
   1 mM CaCl₂
   0.2% NP-40
   
5. MNase resuspension buffer (stored at 4 °C)
   10 mM HEPES-KOH (pH 7.6)
   50 mM KCl
   1.5 mM MgCl₂
   0.5 mM EGTA
   10% glycerol
   
6. Stop buffer (stored at RT)
   2% SDS
   100 mM EDTA (pH 8)
   
7. TE buffer (stored at 4 °C)
   10 mM Tris-HCl (pH 8)
   1 mM EDTA
   

Acknowledgements

This protocol was used for the work previously published in PLoS Genetics (Sancar et al., 2015). This work was supported by grants of the Deutsche Forschungsgemeinschaft: GRK1188 and SFB1036.

References

1. Sancar, C., Ha, N., Yilmaz, R., Tesorero, R., Fisher, T., Brunner, M. and Sancar, G. (2015). Combinatorial control of light induced chromatin remodeling and gene activation in Neurospora. PLoS Genet 11(3): e1005105.
   
2. Vogel, H. J. (1956). A convenient growth medium for Neurospora (Medium N). Microbiol Genet Bull 13:42-43.
   
3. Vogel, H. J. (1964). Distribution of lysine pathways among fungi: Evolutionary implications. Am Nat 98: 435-446.