Dictyostelium ChIP (Chromatin Immuno-precipitation) protocol

Contributed by Jeff Williams, Y. Yamada, and with grateful thanks to Jonathan Chubb for his advice when establishing the method, September 2005.

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[MATERIALS] - [INDEX]

Procedure

Fixation

1. Suspend about 5x10⁸ cells in KK2 to 3.1ml. Add 0.4 ml of 10xPBS and 0.5 ml of 8% paraformaldehyde.
   Rotate for 2 hours at 4°C.
2. 
   
3. Add glycine to 125 mM (0.25 ml of 2M), agitate at room temperature for 5 min.
4. 
   
5. Wash with PBS 4 times and with TBS once. (Can freeze at -80°C here.)
6. 
   

Lysis and sonication

1. Suspend cells in TBS. Add the same volume of 2xTTS/1xTBS and protease inhibitors.
   Mix and leave on ice for 15-20 min.
2. 
   
3. Sonicate DNA down to 0.3-2.5 kb. (9 sec, 5 times with Branson sonifier, output 6)
4. 
   
5. Centrifuge at 14k rpm for 10 min. Transfer sup into a fresh tube and spin again for 20 min. Take sup.
6. 
   

IP and DNA prep

1. Preabsorption:
   
   • Dilute cell lysate twice with TTS buffer.
   • Add protein A Sepharose CL-4B (40 µl of 1:3 slurry) and incubate at 4°C for a few hours.
2. 
   
3. Centrifuge at a top speed for 5 min. Take sup. Keep an aliquot (20 µl) as an input fraction.
4. 
   
5. To cell lysate (1x10⁷ cells, or 400 ng protein) add 40 µl of 10% Blocking Reagent, protease inhibitors and top up with TTS buffer to 0.8 ml.
6. 
   
7. Add antibody and incubate at 4°C overnight.
8. 
   
9. Add 80 µl of 1:3 slurry of Protein A Sepharose CL-4B and incubate at 4°C for several hours.
10. 
    
11. Wash with TTS, 4 times (Spin at a low speed (6000-7000rpm), 1min.
12. 
    
13. Wash with LiDN buffer, 2 times.
14. 
    
15. Spin at a top speed for 1 min and remove the remaining sup.
16. 
    
17. Add 100 µl of E-buffer. Incubate at 37°C for 15 min.
18. 
    
19. Spin and take 90 µl of sup.
20. 
    
21. Incubate at 65°C overnight to reverse crosslink. To input fraction, add E-buffer and do the same.
22. 
    
23. Add 90 µl TE containing Proteinase K (200 mg/ml) and RNase (20 mg/ml). Incubate at 37°C for 1 hour.
24. 
    
25. Purify DNA with QIAquick PCR purification column. Elute with 40-50 µl EB.
26. 
    
27. Analyse by PCR.
28. 
    

[TOP] [INDEX]

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Materials

• 2xTTS/1xTBS
  
  • 50 mM Tris pH8.0
  • 150 mM NaCl
  • 2% Triton X100
  • 4 mM EDTA
  • 0.1% SDS
  • 
    
  
  
• TTS
  
  • 50 mM Tris pH 8.0
  • 150 mM NaCl
  • 1% Triton X100
  • 2 mM EDTA
  • 0.05% SDS
  • 
    
  
  
• LiDN buffer
  
  • 10 mM Tris pH8.0
  • 250 mM LiCl
  • 1% DOC
  • 1% NP-40
  • 1 mM EDTA
  • 
    
  
  
• E-buffer
  
  • 50 mM Tris pH7.5
  • 1% SDS
  • 1 mM EDTA
  • 
    
  
  

[TOP] [INDEX]