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.

[MATERIALS] - [INDEX]

Procedure

Fixation

1. Suspend about 5x10⁸ cells in KK2 to 3.1ml. Add 400 ml of 10xPBS and 500 ml of 8% paraformaldehyde.
   Rotate for 2 hours at 4°C.


2. Add glycine to 125 mM (250 ml of 2M), agitate at room temperature for 5 min.


3. Wash with PBS 4 times and with TBS once. (Can freeze at -80°C here.)

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. Sonicate DNA down to 0.3-2.5 kb. (9 sec, 5 times with Branson sonifier, output 6)


3. Centrifuge at 14k rpm for 10 min. Transfer sup into a fresh tube and spin again for 20 min. Take sup.

IP and DNA prep

1. Preabsorption:
   
   • Dilute cell lysate twice with TTS buffer.
   • Add protein A Sepharose CL-4B (40 ml of 1:3 slurry) and incubate at 4°C for a few hours.
   
   
2. Centrifuge at a top speed for 5 min. Take sup. Keep an aliquot (20 ml) as an input fraction.


3. To cell lysate (1x10⁷ cells, or 400 ng protein) add 40 ml of 10% Blocking Reagent, protease inhibitors and top up with TTS buffer to 0.8 ml.


4. Add antibody and incubate at 4°C overnight.


5. Add 80 ml of 1:3 slurry of Protein A Sepharose CL-4B and incubate at 4°C for several hours.


6. Wash with TTS, 4 times (Spin at a low speed (6000-7000rpm), 1min.


7. Wash with LiDN buffer, 2 times.


8. Spin at a top speed for 1 min and remove the remaining sup.


9. Add 100 ml of E-buffer. Incubate at 37°C for 15 min.


10. Spin and take 90 ml of sup.


11. Incubate at 65°C overnight to reverse crosslink. To input fraction, add E-buffer and do the same.


12. Add 90 ml TE containing Proteinase K (200 mg/ml) and RNase (20 mg/ml). Incubate at 37°C for 1 hour.


13. Purify DNA with QIAquick PCR purification column. Elute with 40-50 ml EB.


14. Analyse by PCR.

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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
  
  
  
  

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