Chromatin Immunoprecipitation (ChIP)

Chromatin Prep

  1. Grow 250 ml cultures to an O.D.(600) = 0.7 - 1.0.

  2. Harvest cells by centrifugation in Sorvall for 5 minutes at 5000 rpm.

  3. Resuspend cells in 50 ml distilled H2O and transfer to a 50 ml conical tube.

  4. Harvest cells by centrifugation at 2000 rpm on the table top centriguge.

  5. Resuspend cells in 10 ml PBS.

  6. (OPTIONAL) Add DMS to a final concentration of 10 mM (100 ul of a 1 M stock in DMSO), incubate 45 minutes at 25oC.

  7. To each 10 ml sample, add 270 ul 37% Formaldehyde so that the final concentration is 1%. Incubate 15 minutes at 25oC.

  8. To each sample, add 0.5 ml 2.5 M Glycine (final concentration is 0.125M). Incubate 5 minutes at 25oC.

  9. Resuspend in 5 ml Breaking buffer (+ Protease Inhibitors) and add an equal volume of glass beads.

  10. Vortex at 25oC for 5 minutes on the multi-vortexer set to level seven (setting depends on the vortexer).

  11. Pierce bottom of 15 ml conical tube with needle, place into a 50 ml conical tube with a Qiagen ring.

  12. Transfer sample (beads and all) into column, and collect lysate by centrifugation at 2000 rpm on the table top centrifuge.

  13. Transfer lysate to 2.0 ml eppendorf tube and pellet chromatin by centrifugation at 14,000 rpm for 2 minutes. Repeat until all lysate has been placed into a single eppendorf tube.

  14. Resuspend extracts in 700 ul ChIP Lysis buffer and sonicate 6-8x15 sec at output 5-6 with tip of probe close to bottom of tube. Avoid foam formation! (Branson, microtip probe).

  15. Cool in ice water after each pulse.

  16. Pellet debris by centrifugation for 5 minutes at 14000 rpm at 4oC to pellet debris.

  17. Transfer supernatant (soluble chromatin) to new 2.0 ml eppendorf tube.

  18. Go to IP reaction or freeze chromatin in liquid N2 and store at -80oC.

Beads and Antibodies

  1. Pipet 20 ul of Protein G Dynabeads per IP in a siliconized tube.

  2. Concentrate the beads on a magnetic particle concentrator (MPC) and remove superntant (do not leave beads on MPC for more than 2 minutes).

  3. Wash beads 3x with 5 mg/ml BSA in PBS (PBSB, freshly prepared).

  4. Add 100 ul per IP of PBSB, resuspend beads well, add antibody (e.g. 1 ul anti-FLAG M2, 2-5 ul anti-Sir2p (goat), 150 ul anti-Sir3p MoAb superntant).

  5. Rotate 2-20 hours at 4oC to bind.

  6. Wash beads 3x with PBSB.

  7. Resuspend beads in 30 ul PBSB/IP and keep on ice until ready to use.

Immunoprecipitation

  1. Thaw chromatin. Take 220 ul per IP.

  2. Spin chromatin for minutes at 14000 rpm at 4oC. Filter supernatant through Millipore Ultrafree-MC 0.45 um unit (1 minute centrifugation at 14000 rpm).

  3. Save 20 ul for input DNA control.

  4. Add 200 ul of chromatin to 20 ul of Protein G Dynabeads (or Protein A beads) as prepared above in siliconized tubes.

  5. Rotate overnight at 4oC to bind.

  6. Concentrate beads on MPC and aspirate off the supernatant.

  7. Wash immunoprecipitations twice in the following buffers in the following order:

    1. 1 ml ChIP lysis buffer
    2. 1 ml ChIP lysis buffer (high salt)
    3. 1 ml ChIP wash buffer
    4. 1 ml TE

  8. For each wash, incubate for 5 minutes at 25oC, then concentrate on MPC and aspirate off the buffer (NOTE: in TE the beads do not stick well to the wall of the tube on the magnet, be careful!!).

  9. When finished with the washes, add 100 ul elution buffer to the beads, and incubate for 10 minutes at 65oC.

  10. Pellet beads by centrifugation for 1 minute at 14000 rpm.

  11. Remove 80 ul of the supernatant to a new 1.7 ml eppendorf tube, and add 70 ul TE, 1 ul RNase A (5 mg/ml), and 10 ul Proteinase K (10 mg/ml).

  12. For 20 ul input samples, add 70 ul TE, 1 ul RNaseA, 60 ul elution buffer and 10 ul Proteinase K)

  13. Incubate 1 hour at 50oC and then 6-20 hours at 65oC to reverse cross-links.

  14. Clean up DNA with QIAquick PCR purification kit: add 750 ul PB, load whole sample on column, wash with 800 ul PE, elute with 50 ul EB.

  15. For protein analysis: boil 30 minutes in 50 ul SDS denaturing buffer.

  16. To check the size of the DNA in the input/supernatnat, add 1 ul RNaseA and incubate for 10 minutes before running the gel.

PCR

  1. Use 1-5 ul of IP and 1 ul of a 1:10 - 1:100 dilution of input for the corresponding PCRs.

  2. Make 50 ul reactions as follows:
    • 27.2-31.2 ul distilled H2O
    • 3.4 ul 1M Tris, pH8.5
    • 0.8 ul 1M NH4SO4
    • 0.5 ul 1% Triton X-100
    • 8.3 ul 30% glycerol
    • 1.25 ul Mg(Ac)2
    • 1 ul 10mM dNTPs
    • 10-20 pmoles for each forward primer, usually 0.2 ul of a 100pM stock (do not use more that 6 ul total primer for each PCR)
    • 10-20 pmoles for each reverse primer, usually 0.2 ul of a 100pM stock
    • 0.5 ul Taq DNA Polymerase

  3. PCR cycles as follows:

    94oC for 3 minutes

    94oC for 30 seconds
    55oC for 30 seconds
    72oC for 45 seconds
      ==> 25-35 cycles

    72oC for 7 minutes
    4oC hold

  4. Load 15 ul on 2% agarose/1xTAE gel. Stain with 1 Vistra Green (or EtBr).

  5. Scan gel at Typhoon (400-600V, 100 micron, +3 mm, Vistra Green/Sybr Green default settings)


2.5M GLYCINE

TO MAKE 500ml: 

  • 93.84 g Glycine
  • distilled H2O to 500 ml


PBS

TO MAKE 1L: 

  • 8 g NaCl
  • 0.2 g KCl
  • 1.15 g Na2HPO4-7H2O
  • 0.2 g KH2PO4
  • distilled H2O to 1L


BREAKING BUFFER (100 mM Tris pH 7.9; 20% glycerol)

TO MAKE 50ml: 

  • 2.5 ml 2M Tris pH 7.9
  • 33.3 ml 30% glycerol
  • 14.17 distilled H2O


ChIP LYSIS BUFFER
(50 mM HEPES pH 7.5, 140 mM NaCl, 1 % Triton X-100, 0.1 % deoxycholate, protease inhibitors)

TO MAKE 500ml: 

  • 25 ml 1 M HEPES pH 7.5 stock solution
  • 14.4 ml 4 M NaCl stock solution
  • 50 ml 10% Triton X100 stock solution
  • 2.0ml 0.5M EDTA stock solution
  • 5 ml 10% deoxycholate stock solution
  • protease inhibitors
  • distilled H2O to 500 ml


ChIP LYSIS BUFFER (high salt)
(50 mM HEPES pH 7.5, 500 mM NaCl, 1 % Triton X-100, 0.1 % deoxycholate, protease inhibitors)

TO MAKE 500ml: 

  • 25 ml 1 M HEPES pH 7.5 stock solution
  • 50 ml 4 M NaCl stock solution
  • 50 ml 10 % Triton X100 stock solution
  • 2.0ml 0.5M EDTA stock solution
  • 5 ml 10 % deoxycholate stock solution
  • protease inhibitors
  • distilled H2O to 500 ml


ChIP WASH BUFFER
(10 mM Tris pH 8.0, 250 mM LiCl, 0.5 % NP-40, 0.5 % deoxycholate, 1 mM EDTA)

TO MAKE 500ml: 

  • 5 ml 1 M Tris pH 8.0 stock solution
  • 5.3 g LiCl
  • 25 ml 10% NP-40 stock solution
  • 25 ml 10% deoxycholate stock solution
  • 1 ml 0.5 M EDTA stock solution
  • distilled H2O to 500 ml


ELUTION BUFFER
(50 mM Tris pH 8.0, 1 % SDS, 10 mM EDTA)

TO MAKE 10ml: 

  • 0.5 ml 1 M Tris pH 8.0 stock solution
  • 1 ml 10% SDS stock solution
  • 0.2 ml 0.5 M EDTA stock solution
  • distilled H2O to 10 ml