整个果蝇胚胎或幼虫成虫盘的染色质免疫沉淀实验

Vincent Loubiere; Anna Delest; Bernd Schuettengruber; Anne-Marie Martinez; Giacomo Cavalli

doi:10.21769/BioProtoc.2327

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Peer-reviewed

Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs

整个果蝇胚胎或幼虫成虫盘的染色质免疫沉淀实验

VL Vincent Loubiere

AD Anna Delest

BS Bernd Schuettengruber

AM Anne-Marie Martinez email

Giacomo Cavalli email

发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2327 浏览次数: 12869

评审: Jihyun KimLeonardo G. GuilgurAnonymous reviewer(s)

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Abstract

Chromatin Immunoprecipitation coupled either to qPCR (qChIP) or high-throughput sequencing (ChIP-Seq) has been extensively used in the last decades to identify the DNA binding sites of transcription factors or the localization of various histone marks along the genome. The ChIP experiment generally includes 7 steps: collection of biological samples (A), cross-linking proteins to DNA (B), chromatin isolation and fragmentation by sonication (C), sonication test (D), immunoprecipitation with antibodies against the protein or the histone mark of interest (E), DNA recovery (E), identification of factor-associated DNA sequences by PCR or sequencing (F). The protocol described here can readily be used for ChIP-seq and ChIP-qPCR experiments. The entire procedure, describing experimental setup conditions to optimize assays in intact Drosophila tissues, can be completed within four days.

Keywords: ChIP (ChIP)

Drosophila (果蝇)

Embryo (胚胎)

Imaginal disc (成虫盘)

Epigenetic mark (表观遗传标记)

Transcription factor (转录因子)

Background

Despite the fact that immortalized cultured cells are extensively used to study the chromatin landscape of various cell types, valuable methods for probing interaction in vivo, under physiological conditions, are necessary to perform temporal or spatial comparative analysis of transcription factor and histone modification maps between different stages of Drosophila development or between different tissues. Here we present a detailed ChIP protocol that has been optimized to work on whole Drosophila embryos and larval imaginal discs, highlighting critical experimental parameters.

Materials and Reagents

Imaginal discs dissection step
1.Sterile tips for micropipettes, 1,000 µl
2.1.5 ml DNA low binding tubes (Eppendorf, catalog number: 022431021 )
3.Schneider’s insect medium (Sigma-Aldrich, catalog number: S0146 )
Embryos collection step
1. Petri dishes
2. 1.5 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 022431021 )
3. Drosophila embryos
4. SAF-INSTANT yeast (Lesaffre)
5. Liquid nitrogen
6. Agar
7. 10% Moldex (Methyl-4-hydroxybenzoate) (Sigma-Aldrich, catalog number: W271004 ) dissolved in water
8. Neutral red (Sigma-Aldrich, catalog number: N4638 )
9. Egg laying medium (see Recipes)
Chromatin immunoprecipitation protocol
1. 1.5 ml DNA low binding (LoBind) tubes (Eppendorf, catalog number: 022431021 )
2. 15 ml sterile plastic tubes (Greiner Bio One International, catalog number: 188261 )
3. 50 ml sterile plastic tubes (Greiner Bio One International, catalog number: 227261 )
4. 15 ml polystyrene Falcon tubes (Corning, catalog number: 352095 )
5. Sterile filter tips, 10 µl, 200 µl, 1,000 µl (STARLAB INTERNATIONAL, catalog numbers: S1121-2710 , S1120-8810 , S1122-1830 ) (see Note 1 for more details)
6. Sterile, DNase and RNase free, DNA low binding filter tips, 10 µl, 200 µl, 1,000 µl (Sorenson BioScience, catalog numbers: 35210 , 35240 , 35260 ) (see Note 1 for more details)
7. Qubit^® Assay tubes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: Q32856 )
8. LightCycler^® 480 multiwell plate 96, white (Roche Molecular Systems, catalog number: 04729692001 )
9. LightCycler^® 480 sealing foil (Roche Molecular Systems, catalog number: 04729757001 )
10. Liquid nitrogen
11. 20% sodium dodecyl sulfate (SDS) (Biosolve, catalog number: 198123 )
12. Autoclaved 1 M KCl solution
13. Autoclaved 5 M NaCl solution
14. Autoclaved 1 M MgCl₂ solution
15. Sterile filtered 1 M HEPES buffer pH 7.6 (Sigma-Aldrich, catalog number: H0887 )
16. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
17. 1 M DL-Dithiothreitol (DTT) prepared from powder (Sigma-Aldrich, catalog number: D0632 ) dissolved in deionized water
18. 1 M sodium butyrate prepared from powder (Sigma-Aldrich, catalog number: 303410 ) dissolved in deionized water
19. EDTA-free protease inhibitor cocktail tablets (Roche Molecular Systems, catalog number: 04693132001 )
20. 16% formaldehyde solution, methanol-free (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 28906 )
21. 2.5 M glycine prepared from powder (Sigma-Aldrich, catalog number: G8898 ) dissolved in deionized water filtered with a disposable syringe filter (0.45 μm) (Pall, catalog number: 4422 )
22. 0.5 M EDTA pH 8.0 (Sigma-Aldrich, catalog number: 03690 )
23. 0.5 M EGTA pH 8.0 prepared from powder (Sigma-Aldrich, catalog number: E3889 ) dissolved in deionized water
24. 1 M sodium deoxycholate prepared from powder (Sigma-Aldrich, catalog number: D5670 ) dissolved in deionized water
25. 20% N-Lauroylsarcosine sodium salt solution (Sigma-Aldrich, catalog number: L7414 )
26. 10 mg/ml RNase A (Sigma-Aldrich, catalog number: R6513 ) dissolved in deionized water
27. UltraPure^TM phenol:chloroform:isoamyl alcohol (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15593049 ) referred as phenol-chloroform in the text
28. 3 M sodium acetate, pH 5.5 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9740 )
29. 35 mg/ml glycogen (MP-Biomedicals, catalog number: 11GLYCO001 )
30. Ethanol absolute AnalaR NORMAPUR^®(VWR, catalog number: 20821.310 )
31. Agarose powder (Sigma-Aldrich, catalog number: A9539 )
32. 1x TAE buffer prepared from 50x solution (Biosolve, catalog number: 205023 )
33. Autoclaved Tris HCl pH 8.0
34. Ethidium bromide (Sigma-Aldrich, catalog number: E1510 )
35. Nuclease free water, not DEPC treated (Thermo Fisher Scientific, Invitrogen^TM, catalog number: AM9937 )
36. Urea (MP Biomedicals, catalog number: 821519 )
37. Sucrose (Sigma-Aldrich, catalog number: S0389 )
38. Bromophenol blue (Sigma-Aldrich, catalog number: B5525 )
39. 100 bp DNA ladder (New England Biolabs, catalog number: N3231S )
40. Qubit^® dsDNA HS Assay Kit (Thermo Fisher Scientific, Invitrogen^TM, catalog number: Q32854 )
41. Dynabeads^® Protein A for Immunoprecipitation (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10001D ) or Dynabeads^® Protein G for Immunoprecipitation (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 10003D ) (see Note 2 for details)
42. 20 mg/ml proteinase K (Sigma-Aldrich, catalog number: P2308 )
43. 1 g/µl of normal rabbit IgG (Cell Signaling Technology, catalog number: 2729 )
44. Sterile filtered water (Sigma-Aldrich, catalog number: W3500 )
45. LightCycler^® 480 SYBR Green I Master (Roche Molecular Systems, catalog number: 04707516001 )
46. 10x embryo buffer stock solution (see Recipes)
47. Embryo buffer (see Recipes)
48. Buffer A1 (see Recipes)
49. Lysis buffer 1 (see Recipes)
50. Lysis buffer 2 (see Recipes)
51. Lysis buffer wash (see Recipes)
52. TE buffer
53. Low blue loading buffer (see Recipes)
54. Elution buffer 1 (see Recipes)
55. Elution buffer 2 (see Recipes)

Equipment

Sieves (pores < 0.5 mm)
Microwave
1 L beaker (Corning, PYREX^®, catalog number: 1003-1L )
Magnetic stirrer
Cages (Dutscher Scientific, catalog number: 789092 )
Wash bottle
Paintbrush (Carolina Biological Supply, catalog number: 173094 )
Calibration checked pipettes 0-2 µl, 1-10 µl, 20-200 µl, 100-1,000 µl
Rotating wheels, one in a cold room and the other one at room temperature
Stereomicroscope
Fine #55 forceps for dissection (Fine Science Tools, catalog number: 11255-20 )
Wheaton^TM Tenbroeck tissue grinder (WHEATON, catalog number: 357422 )
Agarose gel caster
Shaker (For instance the Orbital Shaker from Starlab group) (STARLAB INTERNATIONAL, model: Orbital^TMShaker, catalog number: N2400-8030 )
Cold centrifuge (4 °C, able to reach at least 14,000 x g)
Cold sonicator (we recommend the Bioruptor^® from Diagenode, model: Bioruptor^® Plus sonication device , catalog number: B01020001)
Magnetic rack (Thermo Fisher Scientific, model: DynaMag^TM-2 Magnet, catalog number: 12321D )
Gel visualization system (UV lamp)
Qubit^®fluorometer (Thermo Fisher Scientific, Invitrogen^TM, model: Qubit^® 3.0 , catalog number: Q33216)
LightCycler^® Instrument II (Roche Molecular Systems, model: LightCycler^® 480 Instrument II , catalog number: 05015278001)

Procedure

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Loubiere, V., Delest, A., Schuettengruber, B., Martinez, A. and Cavalli, G. (2017). Chromatin Immunoprecipitation Experiments from Whole Drosophila Embryos or Larval Imaginal Discs. Bio-protocol 7(11): e2327. DOI: 10.21769/BioProtoc.2327.