ChIP

This protocol is in development.~ cmc 15:03, 21 August 2006 (EDT)

Overview

Replace this sentence with a brief description of the protocol and its goal.

Materials

11% Formaldehyde Solution

per 50 ml: (Final concentration)

• 14.9 ml of 37% Formaldehyde (11%)
  
• 1 ml of 5M NaCl (0.1M)
  
• 100 µl of 0.5 M EDTA, pH 8 (1 mM)
  
• 50 µl of 0.5 M EGTA, pH 8 (0.5 mM)
  
• 2.5 ml of 1M Hepes, pH 8 (50 mM)
  

2.5 M glycine (187.5 g/L)

• Dissolve glycine in water with constant stirring
  
• Don’t adjust pH
  

BSA/PBS Solution

per 100 mL:

• 10 mL of 10x PBS
  
• 500 mg of BSA
  
• 90 mL of H20
  

(good for at least a week)

Lysis Buffer 1 (LB1)

per 100 ml: (final concentration)

• 5 ml of 1M Hepes-KOH, pH 7.5 (50 mM)
  
• 2.8 ml of 5M NaCl (140 mM)
  
• 0.2 ml of 0.5M EDTA (1 mM)
  
• 10 ml of glycerol (10%)
  
• 5 ml of 10% NP-40 (0.5%)
  
• 0.25 ml of Triton X-100 (0.25%)
  

Lysis Buffer 2 (LB2)

per 100 ml: (final concentration)

• 4 ml of 5M NaCl (200 mM)
  
• 0.2 ml of 0.5M EDTA (1 mM)
  
• 0.1 ml of 0.5M EGTA (0.5 mM)
  
• 1.0 ml of 1M Tris pH 7.5 (10 mM)
  

Lysis Buffer 3 (LB3)

per 100 ml: (final concentration)

• 0.2 ml of 0.5M EDTA (1 mM)
  
• 0.1 ml of 0.5M EGTA (0.5 mM)
  
• 1.0 ml of 1M Tris-HCl, pH7.5 (10 mM)
  
• 2 mL of 5M NaCl (100 mM)
  
• 1 mL of 10% Na-Deoxycholate (0.1%)
  
• 500 mg of N-lauroyl sarcosine (0.5%)
  

Wash buffer (RIPA buffer)

per 100ml: (final concentration)

• 5ml of 1M Hepes, pH 7.6 (50 mM)
  
• 200µL of 0.5M EDTA (1 mM)
  
• 7 ml of 10% DOC (Na deoxycholate) (0.7%)
  
• 10 ml of 10% NP-40 (IPGEL) (1%)
  
• 10 ml of 5M LiCl or 2.12g powder (0.5 M)
  

Elution buffer

• 50mM Tris pH8
• 1mM EDTA
• 1% SDS

Procedure

I. Cell cross-linking

• Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each ChIP reaction.
  

• Adherent cells:
  1. Add 1/10 volume of fresh 11% formaldehyde solution to plates.
  2. Swirl plates briefly and let them sit at RT for 10 min.
  3. Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde.
  4. Rinse cells twice with 5 ml 1X PBS. Harvest cells using silicon scraper.
  5. Spin cells at 4k for 10’ at 4°C.
  6. Transfer cells to 15ml conical tubes and spin 4k 10’ at 4°C.
  7. Flash freeze cells in liquid nitrogen and store pellets at –80 °C.
• Suspension cells:
  1. Add 1/10 volume of fresh 11% formaldehyde solution to flasks.
  2. Swirl flasks briefly and let them sit at RT for 20 min.
  3. Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde.
  4. Spin cells at 2500rpm for 10’ at 4°C.
  5. Rinse cells with 100ml 1x PBS and spin cells at 2500rpm for 10’ at 4°C.
  6. Rinse cells with 10ml 1x PBS and transfer to 15ml conical tubes and spin 4k 10’ at 4°C.
  7. Flash freeze cells in liquid nitrogen and store pellets at –80 °C.

II. Preblock and binding of antibody to magnetic beads

• Keep beads on ice for all steps

1. wash 100 µL Dynal magnetic beads (per reaction) in 1 ml fresh BSA/PBS
2. collect the beads with magnet wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet.
3. Add 6-10 µg of Ab + 250µL of PBS/BSA.
4. incubate 4 hr to O.N. on a rotating platform at 4∞C.
5. wash beads 3 times in 1.5 ml PBS/BSA.
6. Resuspend in 100µL PBS/BSA.

IV. Chromatin Immunoprecipitation

1. Add 100 µL Ab prebound Dynal magnetic beads from step III.
2. Rock 4∞C 6 hrs to O/N.

Notes

1. This protocol will likely fail the first time you try it. Your LM-PCR product will contain no DNA. Don't loose heart. Try, try again. (Hard to pin down exactly what goes wrong between sonication and LM-PCR, but it seems that the blunting step is very sensitive. Most people get it to work by trying two or three times.) ~ cmc 16:25, 21 August 2006 (EDT)

References

• ChIP protocol, Young Lab

Contacts

• cmc
• odom