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MNase Digestion for Nucleosome Mapping in Neurospora

微球菌核酸酶消化法分析脉孢菌中核小体图谱

CS Cigdem Sancar
GS Gencer Sancar
MB Michael Brunner

发布: 2016年06月05日第6卷第11期 DOI: 10.21769/BioProtoc.1825 浏览次数: 10485

评审: Valentine V TrotterEmily CopeEmilia Krypotou

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Cover of PLOS Genetics, featuring study using the protocol.

Mar 2015

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Abstract

Digestion of chromatin by micrococcal nuclease MNase followed by high throughput sequencing allows us to determine the location and occupancy of nucleosomes on the genome. Here in this protocol we have described optimized conditions of MNase digestion of filamentous fungus Neurospora crassa chromatin without a requirement of a nuclear fractionation step.

Keywords: Neurospora crassa (粗糙脉孢菌) search Nucleosome occupancy (核小体占据) search MNase digestion (MNase消化) search

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 ProbesTM, catalog number: B1595 )
  6. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  7. KCl (Thermo Fisher Scientific, AmbionTM, catalog number: AM9640G )
  8. NaCl (Thermo Fisher Scientific, InvitrogenTM, catalog number: 24740011 )
  9. Tris hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: 10812846001 )
  10. MgCl2 (Thermo Fisher Scientific, AmbionTM, catalog number: AM9530G )
  11. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 793639-500G )
  12. NP-40 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 85124 )
  13. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo FisherTM, 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 FisherTM, catalog number: 17892 )
  19. Glycerol (Sigma-Aldrich, catalog number: G6279 )
  20. Glycogen, molecular biology grade (Thermo Fisher Scientific, Thermo FisherTM, catalog number: R0561 )
  21. Ethanol
  22. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  23. Glycine (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15527013 )
  24. Nuclease free H2O (Thermo Fisher Scientific, Thermo FisherTM, catalog number: R0581 )
  25. Liquid nitrogen
  26. Agarose (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: BP1356100 )
  27. MNase (Sigma-Aldrich, catalog number: N3755-500 )
  28. RNase cocktail enzyme mix (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2286 )
  29. Proteinase K solution (Thermo Fisher Scientific, InvitrogenTM, 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: 05892791001 )
  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 ScientificTM, catalog number: FB966B )
  6. Shaking incubator
  7. Agarose gel electrophoresis

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Sancar, C., Sancar, G. and Brunner, M. (2016). MNase Digestion for Nucleosome Mapping in Neurospora. Bio-protocol 6(11): e1825. DOI: 10.21769/BioProtoc.1825.

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分类

微生物学 > 微生物遗传学 > DNA > 染色体

微生物学 > 微生物生物化学 > DNA

分子生物学 > DNA > DNA-蛋白质相互作用

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