Standard Operating Procedure

 

Chromatin Immunoprecipitation (ChIP)

 

Hazards:  Use Formaldehyde in a fume hood. 

 

Protection:

Wear gloves. Wear ear protection during sonication.  Led Zeppelin is very good to listen to while sonicating, it drowns out the buzzing.

 

Waste:

 

Spill Clean-Up:

 

Notes:  Optimize sonication for your cells before starting.  This is very important and needs to be done for each cell type you will be using.  Cross-linked DNA should be sheared to 200-1000 bp in length.  Sonicate cells in a 1.5 ml eppendorf tube.  Use the Fisher 60 sonic dismembrator in the dishwasing room with the microtip from Dan MuellerÕs lab.  Dip probe just below surface of sample.  To prevent foaming, turn on sonicator once probe has been inserted and turn it off before removing.  If foaming occurs during sonication, spin for 4 minutes at 8000 rpm to remove trapped air.  Keep tube on dry ice for 20 seconds between pulses.  Do not let the sample freeze.  This procedure is for 107 cells per condition.

 

Procedure:

Day 1

  1. Place cells in a 1.5 ml eppendorf tube.  Use 107 cells per condition.
  2. Cross-link cells by adding 37% formaldehyde to medium at a final concentration of 1% formaldehye.  Incubate for 10 minutes at RT with gentle rocking.
  3. Add 1M glycine to a final concentration of 135 mM to quench fixation.
  4. Warm lysis buffers to room temperature.
  5. Add protease inhibitors to Lysis buffers, PBS wash, and ChIP dilution buffer.
    1. 1 µg/ml Aprotinin (stock at 5 mg/ml)
    2. 1 µg/ml Pepstatin (stock at 1 mg/ml)
    3. 1 mM PMSF (stock at 100 mM)  
  6. Wash cells 2X with ice-cold 1X PBS.  (Spin at 4000 rpm, 5 min)
  7. Add 1000 µl ChIP lysis buffer/107 cells.  Incubate on ice for 10 minutes.
  8. Spin at 14000 rpm for 5 minutes to collect nuclei.  Discard sup.
  9. Add 600 µl Nuclear Lysis buffer/107 cells.
  10. Use a 23 gauge needle to disrupt the nuclei.
  11. Incubate on ice, 10 minutes.
  12. Sonicate.  15 pulses for 20 seconds at a power of 4 with a 20 second rest on dry ice between pulses.
  13. Spin at 14000 rpm, 10 minutes, 4¼C to clarify soluble chromatin.
  14. Freeze sonication control at -20¼C.
  15. Transfer sup to new eppendorf tube.
  16. Add 900 µl ChIP dilution buffer for a total of 1.5 ml.
  17. Add 80 µl Protein A sepharose beads for 30 minutes hr at 4¼C with shaking (use shaker in 6-221 cold room) to pre-clear non-specific chromatin.  Cut off end of pipette tips.
  18. Spin 1 min, 1200 rpm.
  19. Transfer sup only (no beads!) to a new eppendorf tube.
  20. Remove 150 µl for input DNA (1/10).  Store at -20¼C.
  21. Divide remaining sup into 2 tubes.  One tube for the negative control (no antibody or isotype antibody).  The other tube is with antibody.  Add ChIP dilution buffer for a total of 1.5 ml/tube.  Use 4 µg of anti-acetyl-H3 antibody.  Incubate overnight at 4¼C with rotation.

 

 

Day2

  1. Add 60 µl Protein A sepharose beads for 2 hr at 4¼C with rotation to bind immune complexes to the beads.
  2. Pellet beads at 1200 rpm for 1 minute.
  3. Carefully remove and discard sup that contains the unbound, non-specific DNA
  4. Wash Protein A sepharose/antibody histone complex for 4 minutes at RT with rotation with 1 ml of the following buffers.  Spin at 1200 rpm for 1 minute between washes.  Discard sup.
    1. Low Salt wash buffer
    2. High Salt wash buffer
    3. LiCl wash buffer
    4. 1X TE:  2 washes
  5. Prepare SDS elution buffer (1% SDS, 0.1 M NaHCO3).  We have 10X stocks, for 2.5 ml, use 250 µl 10% SDS, 250 µl IM NaHCO3, and 2 ml dH20.
  6. Pre-heat dry bath to 65¼C.
  7. Add 250 µl SDS elution buffer to beads.  Vortex for 15 seconds, rotate, 15 minutes, RT.
  8. Spin at 1200 rpm for 1 minute.
  9. Transfer sup to a new eppendorf tube.
  10. Repeat elution (steps 27-29).  (The total volume of the combined elutates = 500 µl.) 
  11. Thaw input and sonication control tubes.
  12. To reverse the cross-links, add 20 µl 5 M NaCl (final concentration of 0.2M) and incubate at 65¼C for 4 hours in a dry bath.  At this point also reverse cross-links of input DNA that has been stored at -20¼C. After 4 hours, add 1 µl 10 mg/ml RNase A, incubate for another hour, for a total of 5 hours.
  13. Add 2 ½ volumes of 100% ethanol, 1/10 volume 3M Na acetate, pH 5.2, and 2 ul yeast tRNA (to visualize pellet).  Precipitate overnight at -20¼C.

 

Day 3

  1. Pre-heat dry bath to 42¼C.
  2. Spin at 14000 rpm for 15 minutes, 4¼C.  Carefully discard sup.
  3. Add 1 ml 70% ethanol.  Spin at 14000 rpm for 2 minutes, RT.
  4. Remove ethanol.
  5. Spin again, remove all ethanol.
  6. Add 100 µl dH20, 2 µl 0.5M EDTA, 4 µl 1 M Tris, pH 6.5, and 2 µl 10 mg/ml Proteinase K, incubate for 1-2 hours at 45¼C in a dry bath.
  7. Apply sample to a QiaQuick spin column.  Spin at 14000 rpm for 1 minute.
  8. Add 50 µl EB buffer to column. Spin at 14000 rpm for 1 minute.
  9. PCR.

 

 

Reagents:

ChIP Lysis Buffer (50 ml)

5 mM PIPES (0.0756 g)

85 mM KCl (0.32 g)

0.5% NP-40 (250 µl)

 

Nuclear Lysis Buffer (50 ml)

50 mM Tris-HCl (2.5 ml 1 M Tris)

10 mM EDTA (1 ml 0.5 M EDTA)

1% SDS (500 µl 10% SDS)

 

Low Salt Wash Buffer (50 ml)

0.1% SDS (500 µl 10% SDS)

1% Triton-X100 (500 µl)

2 mM EDTA (200 µl 0.5 M EDTA)

150 mM NaCl (1.5 ml 5 M NaCl)

20 mM Tris-HCl, pH 8.1 (1 ml 1 M Tris, pH 8.1)

 

High Salt Wash Buffer (50 ml)

0.1% SDS (500 µl 10% SDS)

1% Triton-X100 (500 µl)

2 mM EDTA (200 µl 0.5 M EDTA)

500 mM NaCl (5 ml 5 M NaCl)

20 mM Tris-HCl, pH 8.1 (1 ml 1 M Tris, pH 8.1)

 

LiCl Wash Buffer (50 ml)

0.25 M LiCl (0.53 g)

1% NP-40 (500 µl)

1% deoxycholate (0.5 g)

1 mM EDTA (100 µl 0.5 M EDTA)

10 mM Tris-HCl, pH 8.1 (500 µl 1 M Tris, pH 8.1)

 

ChIP dilution buffer (50 ml)

0.01% SDS (50 µl 10% SDS)

1.1% Triton X-100 (550 µl)

1.2 mM EDTA (120 µl 0.5 M EDTA)

16.7 mM Tris-HCl, pH 8.1 (835 µl 1 M Tris)

167 mM NaCl (1.67 ml 5M NaCl)

 

TE Buffer

10 mM Tris-HCl, pH 8.0

1 mM EDTA