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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

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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), Nucleosome occupancy(核小体占据), MNase digestion(MNase消化)

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 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, Invitrogen™, 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, 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 ScientificTM, 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.

简介

通过微球菌核酸酶MNase消化染色质,然后高通量测序允许我们确定核小体在基因组上的位置和占据。 在这个协议中,我们描述了MNase消化丝状真菌粗糙链孢霉染色体的优化条件,而不需要核分馏步骤。

关键字:粗糙脉孢菌, 核小体占据, MNase消化

材料和试剂

  1. 滤纸(VWR International,目录号:1001-090)
  2. D - (+) - 葡萄糖一水合物(Sigma-Aldrich,目录号:49159)
  3. (Sigma-Aldrich,目录号:608033)代替L-精氨酸 - 13 C 15 - - -
  4. 1伏格尔盐(Vogel,1956; Vogel,1964)
  5. D-生物素(Thermo Fisher Scientific,Molecular Probes TM,目录号:B1595)
  6. 蔗糖(Sigma-Aldrich,目录号:SO389)
  7. KCl(Thermo Fisher Scientific,Ambion TM,目录号:AM9640G)
  8. NaCl(Thermo Fisher Scientific,Invitrogen TM,目录号:24740011)
  9. Tris盐酸盐(Tris-HCl)(Sigma-Aldrich,目录号:10812846001)
  10. MgCl 2(Thermo Fisher Scientific,Ambion TM,目录号:AM9530G)
  11. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:793639-500G)
  12. NP-40(Thermo Fisher Scientific,Thermo Scientific TM,目录号:85124)
  13. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Fisher TM ,目录号:20290)
  14. 亚精胺(Sigma-Aldrich,目录号:S0266)
  15. HEPES(Sigma-Aldrich,目录号:H4034)
  16. EGTA(Sigma-Aldrich,目录号:E3889)
  17. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  18. EDTA(Thermo Fisher Scientific,Thermo Fisher< sup>,目录号:17892)
  19. 甘油(Sigma-Aldrich,目录号:G6279)
  20. 糖原,分子生物学级(Thermo Fisher Scientific,Thermo Fisher TM ,目录号:R0561)
  21. 乙醇
  22. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  23. 甘氨酸(Thermo Fisher Scientific,Invitrogen TM,目录号:15527013)
  24. 核酸酶游离H 2 O(Thermo Fisher Scientific,Thermo Fisher TM ,目录号:R0581)
  25. 液氮
  26. 琼脂糖(Thermo Fisher Scientific,Fisher Scientific ,目录号:BP1356100)
  27. MNase(Sigma-Aldrich,目录号:N3755-500)
  28. RNA酶混合酶混合物(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2286)
  29. 蛋白酶K溶液(Thermo Fisher Scientific,Invitrogen TM,目录号:AM2548)
  30. Wizard SV凝胶和PCR清除系统(Promega Corporation,目录号:A9281)
  31. 无EDTA蛋白酶抑制剂片剂(Roche Diagnostics,目录号:05892791001)
  32. C
  33. ZnSO 4 。 7H O
  34. Fe(NH 4)2子(SO 4)子部分2子部分。 > 2 O
  35. CuSO 4 5H sub 2 O
  36. MnSO 4 1 H sub 2 O
  37. (无水)
  38. Na 2 MoO 4 sub 。 2H O
  39. 氯仿
  40. MilliQ水
  41. Na 3 6 7
  42. KH 2 PO 4
  43. NH 4 3
  44. MgSO 4。 。 O
  45. CaCl 2 2H O
  46. 液体介质(见配方)
  47. 微量元素溶液(参见配方)
  48. 50x Vogel盐(见配方)
  49. 裂解缓冲液(见配方)
  50. MNase重悬缓冲液(参见配方)
  51. 停止缓冲区(参见配方)
  52. TE缓冲区(参见配方)

设备

  1. 离心机
  2. 涡流
  3. 砂浆和杵
  4. 热混合器(Eppendorf AG,型号:Thermo-mixer R)
  5. Buhner漏斗(Thermo Fisher Scientific,Fisher Scientific ,目录号:FB966B)
  6. 摇动培养箱
  7. 琼脂糖凝胶电泳

程序

  1. 在500ml烧瓶中将约2×10 8个粗糙脉孢菌分生孢子接种到200ml液体培养基中。
  2. 在所需光照和温度条件下以100rpm振荡培养培养物。代表性培养物在24℃和80μE光强度下生长三天。
  3. 为了交联,向培养物中加入多聚甲醛至终浓度为1%,并在24℃下振荡孵育10分钟。
  4. 淬灭多聚甲醛5分钟,用甘氨酸终浓度为125mM。
  5. 通过布氏漏斗过滤液体培养基,并用500ml水(室温)冲洗菌丝体。在纸巾之间挤压菌丝体以除去所有的水,然后将菌丝体放在Eppendorf管中,立即在液氮中冷冻。在该步骤中,可将菌丝体储存在-80℃。
  6. 冷却研钵和杵通过浸没在液氮中5分钟。取出研钵和杵,然后立即将冷冻的菌丝体放入研钵中,用杵研磨菌丝体,直到它是细粉碎的粉末。在这一步应避免温暖菌丝体/地面菌丝体粉末。
  7. 将重新悬浮〜3.75mg裂解缓冲液中的〜400mg研磨菌丝体与新添加的0.5mM DTT,0.5mM亚精胺和无EDTA蛋白酶抑制剂片剂。
  8. 通过涡旋混合悬浮液,并在冰上孵育5分钟
  9. 将悬浮液分配到五个1.5毫升的Eppendorf管(每管750微升)。
  10. 将MNase粉末重悬在850μlMNase重悬缓冲液中(估计浓度= 0.58 U /μl)。 等分试样可以储存在-20℃。 避免冻融循环。
  11. 向每个管分别加入0 [对照],0.75,1.5,3,6单位的MNase。 将所有样品在25℃下孵育1小时,同时在热混合器中以400rpm振摇
  12. 向每个样品中加入85μl终止缓冲液以停止消化,并以20,000×g离心20分钟以沉淀细胞碎片。 将上清转移到新管
  13. 向每个上清液中加入2μlRNA酶混合酶混合物,并在Thermo-mixer中在37℃下以400rpm振荡孵育45分钟。
  14. 向每个样品中加入15μl的蛋白酶K溶液,并在65℃下在热混合器中以400rpm振荡2小时。
  15. 向每个样品中加入40μg糖原和1,250μl的100%EtOH,并在-20℃下孵育30分钟。
  16. 在4℃下以最大速度(〜20,000×g/g)离心30分钟。 弃去上清液。 用700μl70%冰冷的乙醇洗涤DNA沉淀。 在不打扰沉淀的情况下缓慢地移除乙醇。 不需要干燥颗粒。
  17. 将洗涤过的DNA沉淀溶解在100μl无DNAse/RNA酶的H 2 O中。
  18. 通过使用PCR清除试剂盒进一步纯化DNA样品。
  19. 加载样品在1.7%琼脂糖凝胶(80伏1小时),以分析核小体梯(图1)。

代表数据


图1. MNase消化和纯化的 脉孢菌 核糖体DNA装载在1.7%琼脂糖凝胶上的代表性图像

食谱

  1. 液体介质(新鲜制备)
    2%(w/v)葡萄糖单水合物0.5%(w/v)L-精氨酸 1伏格尔盐(Vogel,1956; Vogel,1964)
    10 ng/ml生物素
  2. 微量元素溶液(在RT保存)
    将所示量溶于95ml MilliQ水中
    5克C 6 H 8 H 8 O 7
    5g ZnSO 4 7H 2 O 1 g Fe(NH 4)2(SO 4)2 sub 2+。 2 O
    0.25g CuSO 4 5H O 0.05g MnSO 4 sub 。 1H 2 O 0.05g H 3 BO 3(无水)
    0.05g Na 2 SO 4 MoO 4 sub。 2H 2 加入1ml氯仿作为防腐剂
  3. 1 L 50x Vogel盐(在RT保存)
    将所示量溶于755ml MilliQ水中
    125g Na 3 H 6 CH 6 H 5 O 7 7 / 250克KH 2 PO 4 sub/
    100g NH 4 NO 3 sub/
    10g MgSO 4·7H 2 O 7H/SiO 2 5g CaCl 2 2H 2 5ml微量元素溶液
    250μg生物素
    无需调整pH值
    加入5ml氯仿作为防腐剂
  4. 裂解缓冲液(储存在4℃) 250mM蔗糖 60 mM KCl
    15mM NaCl 15mM Tris-HCl(pH7.4) 3mM MgCl 2/
    1mM CaCl 2
    0.2%NP-40
  5. MNase重悬缓冲液(储存于4℃) 10mM HEPES-KOH(pH7.6) 50 mM KCl
    1.5mM MgCl 2·h/v 0.5mM EGTA 10%甘油
  6. 停止缓冲区(在RT存储)
    2%SDS
    100mM EDTA(pH8)
  7. TE缓冲液(储存在4℃) 10mM Tris-HCl(pH8)
    1mM EDTA

致谢

该方案用于以前在PLoS Genetics(Sancar等人,2015)中发表的工作。 这项工作得到德意志研究所授予的资助:GRK1188和SFB1036。

参考文献

  1. Sancar,C.,Ha,N.,Yilmaz,R.,Tesorero,R.,Fisher,T.,Brunner,M。和Sancar,G。 组织控制光诱导的染色质重塑和基因在细胞孢子菌中的激活。 PLoS Genet 11(3):e1005105。
  2. Vogel,H.J。(1956)。 用于孢子菌的方便的生长培养基(培养基N)。 Microbiol Genet Bull 13:42-43。
  3. Vogel,H.J。(1964)。 真菌间赖氨酸途径的分布:进化影响。 98:435-446。
  • English
  • 中文翻译
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. 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.
  2. 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.
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