ChIP

Reagents:

 

ChIP sonication Buffer

0.5 g 1% SDS (precipitates out when cold)

1 ml 0.5 M EDTA

2.5 ml 1 M Tris-Cl pH 8.1

50 ml of ddH2O

 

(Alternate Wash Buffer) High Salt Wash Buffer (1% Triton X-100, 0.1% Deoxycholate, 50 mM Tris-8.1, 500 mM NaCl, 5 mM EDTA)

10 ml 10% Triton X-100

1 ml 10% Deoxycholate

5 ml 1M Tris-8.1

1 ml 0.5M EDTA

10 ml 5M NaCl

73 ml Water

 

(Alternate wash buffer) LiCl Immune Complex Wash Buffer

25 ml 1M LiCl

5 ml 10% IGEPAL

5 ml 10% Deoxycholate

1 ml 1M Tris-8.1

200 ul 0.5M EDTA

64 ml Water

 

1X TE Buffer (10mM Tris, 8.1, 1 mM EDTA)

 

0.5 M EDTA

 
1 M Tris-Cl, pH 6.8
 

Protein A/G Agarose (Santa Cruz #sc-2003)

 (in the fridge with the antibodies)

 

Proteinase K (19 mg/ml, Boehringer Mannheim # 1964372)

(radiation fridge)

 

10X proteinase K buffer (25 ml)

3.125 ml 1 M Tris (PH 8.0)

3.125 ml 0.5 M EDTA (PH 8.0)

25ml of ddH2O 

 

Protease inhibitor cocktail (-20 degree fridge)

 

Elution Buffer (1% SDS, 0.1 M NaHCO3, 0.01 mg/ml Herring sperm DNA)

 

10 mg/ml Salmon Sperm DNA

 

37% Formaldehyde (ACS reagent grade)

 

1.25 M glycine

 

100X BSA

10 µg BSA/ml ddH2O

 

 

Protocol: (Generalized for all cell types- inquire for details regarding particular cell types)

 

For all the following steps, use the pipets that are specifically designated for ChIP use only and the filter pipette tips.

 

PART 1 (Whole Cell Extract WCE)

  1. To each 10 cm dish of cells (3 plates 80%-90% confluent), wash plate once with 10ml of PBS, then add 10 ml of Fresh PBS and add 270 ul of 37% formaldehyde, swirl gently to mix, and place at room temp 10 min.
  2. At the end of the incubation, add 1 ml of 1.25 M glycine, swirl to mix.
  3. Aspirate medium (ASAP after adding glycine or cells will detach)
  4. wash plate with 10 ml cold PBS x 2.  Aspirate PBS completely after the second wash.
  5. add 500 ul of cold PBS + 6 µl protease inhibitors per plate and scrape cells, collect in a 1.5 ml centrifuge tube.  At this point you should pool three plates worth of cells together into 2 tubes.
  6. centrifuge at 2000 rpm for 2 min at 4 º C.
  7. remove and discard PBS
  8. add 600 ul of ChIP sonication buffer + 6 µl of protease inhibitors per tube, and resuspend pellet (you can vortex vigorously at this point). 
  9. Place on ice for 10 min.
  10. Sonicate at a setting of 40% Amp for 15 pulses (possibly fewer, but 15 will ensure sufficient shearing of the DNA), 10 seconds per pulse for 2 min. 30 sec (pulse time), (placing the cells on an ice-water bath between pulses).  To eliminate foaming, place the tip of the horn near the bottom of the tube while touching the side with the horn. Do this step in the cold room and sonicate while on ice, because they will heat up.
  11. Centrifuge at maximal setting at 4 C for 10-15 min.
  12. Remove the supernatant into a fresh tube.  This is the Whole Cell Extract (WCE), and can be stored at –80 C at this point, if desired.

 

PART 2

  1. Add a sufficient amount of ChIP buffer to perform the immunoprecipitations.  A final volume of 1.5 ml is usually good.  Add protease inhibitors to this (about 6 ul of protease inhibitor cocktail).
  2.  Add 1 µl of salmon sperm DNA before splitting the samples. Also add 10 µl of 100x BSA.  Mix well.
  3.  Split the samples into (+) and (-) antibody samples of equal volume, making sure you choose an amount that will allow you to withhold 10% of the IP sample volume for your  “10% input”  samples that you need later for PCR, as well as having a little leftover sample in case you want to examine them later.  For example, if you have 1 ml after step 13, split it into a 400 µl (+) sample, a 400µl (-) sample, 400 µl isotype sample, a 40 µl input sample,  with 160 µl leftover.
  4. Label and freeze the 10% Input samples in -20º and leftovers in -80º.
  5. To the (+) antibody sample, add 5 ul of target protein antibody.  This amount may vary according to the antibody used and the size of your sample.  To the (-) antibody sample, add nothing. Add 5µl of IGG to the Isotype control.
  6. Place the samples on a nutator in the cold room, and rotate overnight.  This step could also be done only for 2hrs in the cold room.

 

PART 3

  1. Resuspend Protein A/G agarose so that it forms a uniform suspension.  Using a pipet tip with the end clipped off, add 40 ul of this suspension to each immunoprecipitation.  Resuspend the protein A/G  agarose each time before adding to the next sample, as it settles quickly.
  2. Add 2 ul of a 10 mg/ml solution of salmon sperm DNA
  3. Place back on the nutator at 4 C for 1-2 h.
  4. Centrifuge the samples at 4 C for 1 min at 2500 rpm.
  5. Carefully remove the supernatant using a P-1000 and place it in a tube and label it “sample X—sup.”  Place this at –20 C in case you need it later.
  6. Add 1 ml of COLD ChIP buffer (no protease inhibitors), invert the sample to resuspend the resin, and centrifuge for 1 min. at 2500 rpm.
  7. remove and discard the supernatant.
  8. Wash 2x in 1 ml cold 1X PBS, spinning as above and discarding the supernatants. Note- wash buffer may vary according to conditions, antibody used.  See alternate wash buffers at top of protocol. (order of alternate wash buffers after PBS: High salt, then Li-Cl)
  9. Add 250 ul of Elution buffer to the resin, and place on a nutator at room temp. for 15-20 min.
  10. Centrifuge at top speed 60s to pellet the resin, remove the supernatant to a fresh tube.
  11. Repeat the elution step (step 9), except that it is recommended to place tubes in a 100C heat block for 60s before placing on the nutator.
  12. Spin as in step 10 and combine with the supernatants from step 10. (you can now discard the tube containing the resin pellet)
  13. At this time, add 500 µl of elution buffer to the “10% input samples” from step 4 (PART 2).  Process them along with your other samples from here on.
  14. Add 20 µl of 5 M NaCl to each sample, vortex to mix, and place in a 65 C thermomixer for 3-4 h.
  15. Add 1 ml of ROOM TEMP ethanol to each sample place at –20 C overnight.

 

PART 4

  1. Next day, spin the samples at top speed at 4 C for 15-20 min. to pellet the precipitated protein/DNA.  Be sure to pre-chill the centrifuge.
  2. Aspirate off the supernatant, add 1 ml of ice cold 70% ethanol, spin again at 4 C for 5 min.
  3. Aspirate off the sup, allow to air dry for 5-10 min.
  4. Dissolve the pellet in 100 µl of TE.
  5. Add 11 µl of 10X Proteinase K buffer, and 1 ul of a 19 mg/ml proteinase K solution.
  6. incubate at 55 C for 1 h in the thermomixer.
  7. Add 390 µl TE
  8. Extract w/ 500 µl phenol:CHCl3:isoamylalcohol
    1. Add the stuff
    2. Vortex high speed 1 min
    3. Spin high speed 1 min
  9. Remove top (aqueous phase) and put into new tube.
  10. Add 44 µl 3 M NaOAc (sodium acetate) and 1 ml EtOH
  11.  Place at –20C overnight

 

PART 5

  1.   Spin sample at high speed 4C  5 min.
  2.   Aspirate sup.  Add 1 ml ice cold 70% EtOH. Spin again high speed 4C  5 min.
  3. Aspirate sup, air dry pellet about 20 min.
  4.  Resuspend pellets in 100 µl TE (vortex). 
  5. Ready for PCR.
  6.  If samples, especially the inputs, give strange looking or flat curves on the I-cycler, try adding 390 ml TE to the samples and repeating steps 1-6.


Source: http://faculty.virginia.edu/mirmira/resources_files/Protocols/MirmiraLabChIPprotocol.htm (with our personal edits)