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
发布: 2016年06月05日第6卷第11期 DOI: 10.21769/BioProtoc.1825 浏览次数: 9698
评审: Valentine V TrotterEmily CopeEmilia Krypotou
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
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)
Nucleosome occupancy (核小体占据)
MNase digestion (MNase消化)
Materials and Reagents
- Filter paper (VWR International, catalog number: 1001-090 )
- D-(+)-glucose monohydrate (Sigma-Aldrich, catalog number: 49159 )
- L-arginine-13C6,15N4 hydrochloride (Sigma-Aldrich, catalog number: 608033 )
- 1x Vogel’s salt (Vogel, 1956; Vogel, 1964)
- D-biotin (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: B1595 )
- Sucrose (Sigma-Aldrich, catalog number: S0389 )
- KCl (Thermo Fisher Scientific, AmbionTM, catalog number: AM9640G )
- NaCl (Thermo Fisher Scientific, InvitrogenTM, catalog number: 24740011 )
- Tris hydrochloride (Tris-HCl) (Sigma-Aldrich, catalog number: 10812846001 )
- MgCl2 (Thermo Fisher Scientific, AmbionTM, catalog number: AM9530G )
- Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 793639-500G )
- NP-40 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 85124 )
- Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo FisherTM, catalog number: 20290 )
- Spermidine (Sigma-Aldrich, catalog number: S0266 )
- HEPES (Sigma-Aldrich, catalog number: H4034 )
- EGTA (Sigma-Aldrich, catalog number: E3889 )
- Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
- EDTA (Thermo Fisher Scientific, Thermo FisherTM, catalog number: 17892 )
- Glycerol (Sigma-Aldrich, catalog number: G6279 )
- Glycogen, molecular biology grade (Thermo Fisher Scientific, Thermo FisherTM, catalog number: R0561 )
- Ethanol
- Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
- Glycine (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15527013 )
- Nuclease free H2O (Thermo Fisher Scientific, Thermo FisherTM, catalog number: R0581 )
- Liquid nitrogen
- Agarose (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: BP1356100 )
- MNase (Sigma-Aldrich, catalog number: N3755-500 )
- RNase cocktail enzyme mix (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2286 )
- Proteinase K solution (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM2548 )
- Wizard® SV gel and PCR clean-up system (Promega Corporation, catalog number: A9281 )
- EDTA-free protease inhibitor tablets (Roche Diagnostics, catalog number: 05892791001 )
- C6H8O7
- ZnSO4.7H2O
- Fe(NH4)2(SO4)2.6H2O
- CuSO4.5H2O
- MnSO4.1H2O
- H3BO3 (anhydrous)
- Na2MoO4.2H2O
- Chloroform
- MilliQ water
- Na3C6H5O7
- KH2PO4
- NH4NO3
- MgSO4.7H2O
- CaCl2.2H2O
- Liquid medium (see Recipes)
- Trace element solution (see Recipes)
- 50x Vogel’s salt (see Recipes)
- Lysis buffer (see Recipes)
- MNase resuspension buffer (see Recipes)
- Stop buffer (see Recipes)
- TE buffer (see Recipes)
Equipment
- Centrifuge
- Vortex
- Mortar and pestle
- Thermo-mixer (Eppendorf AG, model: Thermo-mixer R )
- Buhner funnel (Thermo Fisher Scientific, Fisher ScientificTM, catalog number: FB966B )
- Shaking incubator
- Agarose gel electrophoresis
Procedure
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
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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