ChIP Supplementary Protocols

Preparing cells for ChIP

  1. If necessary, stimulate or treat adherent mammalian cells at ~80 to 90% confluence in a 150 mm culture dish containing 20 mL of growth media. Include one extra plate of cells to be used solely for estimation of cell number.
  2. Prepare 22 mL of 1X PBS (2.2 mL 10X PBS and 19.8 mL water) for each 150 mm culture dish. Store on ice. This will be used for washes and needs to be ice cold.
  3. Add 550 µL of 37% formaldehyde (or 1100 µL of 18.5% formaldehyde) directly to 20 mL of growth media to crosslink. Gently swirl dish to mix.
  4. Incubate at room temperature for 10 minutes.
  5. During the ten minute incubation, prepare 1X protease inhibitor in PBS: Add 2 mL of ice cold 1X PBS to a separate tube for every dish and add 10µL of the 200X Protease Inhibitor Cocktail III. Store on ice.
  6. Add 2 mL of 10X glycine to each dish to quench excess formaldehyde.
  7. Swirl to mix and incubate at room temperature for 5 minutes.
  8. Place dishes on ice.
  9. Aspirate medium, removing as much medium as possible, being careful not to disturb the cells. If you are using suspension cells, spin down cells at 8000 x g for 5 minutes.
  10. Add 10 mL of cold 1X PBS to wash cells. Remove 1X PBS.
  11. Remove 1X PBS and repeat wash.
  12. Add 2 mL of 1X Protease Inhibitor Cocktail III in PBS prepared in Step 6.
  13. Scrape cells from each dish into a separate microcentrifuge tube.
  14. Spin at 800 x g at 4 °C for 5 minutes to pellet cells.

Preparing tissues for ChIP

  1. Isolate non-fixed fresh tissue as desired. Use  a razor blade to cut a pea-size piece of tissue into small pieces (typically 1mm or smaller) to improve crosslink efficiency. Alternatively, a plug of tissue from cryosectioned non-formalin-fixed- paraffin-embedded (FFPE) material can be used to obtain a small sample of interest (please see the Magna ChIP™ G Tissue Kit manual, Cat. No: 17-20000).
  2. Weigh the tissue, and then transfer into a 50 mL tube and wash twice with ice cold 1X PBS.
  3. Resuspend tissue in 20 mL ice cold PBS and add 550 µL of 37% formaldehyde (or 1100 µL of 18.5% formaldehyde) to crosslink. Gently swirl dish to mix.
  4. Incubate at room temperature for 10 minutes.
  5. In the interim, prepare 1X protease inhibitor in PBS: Add 2 mL of ice-cold 1X PBS to a separate tube for every sample and add 10 µL of Protease Inhibitor Cocktail III. Store on ice.
  6. Add 2 mL of 10X glycine to quench excess formaldehyde.
  7. Homogenize the tissues several times using a Dounce homogenizer (loose pestle).
  8. Spin at 800 x g at 4 °C for 5 minutes to pellet cells.

Optimizing Sonication and Analyzing DNA Fragments

Optimal conditions for shearing crosslinked DNA to 200-1000 base pairs in length depend on the cell type, cell concentration, and the specific sonicator equipment, including the power settings and duration and number of pulses. Approaches for optimizing sonication may include the following:

A. Varying the concentration of cell equivalents per mL of initial buffer  with constant sonication parameters.
B. Choosing a fixed concentration of cell equivalents per mL of buffer and varying cycles and/or power settings  of sonication.
C. A combination of both approaches.

The protocol below describes optimization following option A and is provided as an example only.

  1. Generate a cell lysate by following Section 5.1, but vary your buffer volume per cell amount to generate 3 different microcentrifuge tubes containing several cell equivalent concentrations in the range of 5 x 106 per mL to 5 x 107 per mL. For HeLa cells, this requires approximately 4 x 107 cell equivalents, or approximately four 15 cm plates.
  2. Continue with the following Cell Lysis procedure. Each microcentrifuge tube should contain approximately 500 µL of cell lysate.

 

Volume of Cell lysis Buffer Cell Density Cells required
500 µL 5 x 106/mL 2.5 x 106
500 µL 2 x 107/mL 1 x 107
500 µL 5 x 107/mL 2.5 x 107

  1. Be sure to keep the samples on wet ice at all times. Sonication generates heat which will denature the chromatin.
  2. Remove 1 x 105 cell equivalents from each condition prior to sonication for analysis of unsheared DNA.
  3. For each cell concentration, sonicate each tube for a fixed number of cycles allowing rests between cycles according to the instrument manufacturer’s guidelines. For example, using a Misonix 3000 instrument and a No. 419 microtip probe, use six 15 second pulses with 50 second intervals between pulses, with power setting at 6. Keep tubes cool at all times.
  4. Remove 1 x 105 cell equivalents (20 µL, 5 µL, 2 µL from least to most concentrated sample) of the sonicated chromatin from each condition to a fresh tube.
  5. To all samples (unsheared and sheared), add elution buffer to a final volume of 50 µL.
  6. Add 1 µL Proteinase K and incubate at 62 °C for 2 hour.
  7. Load 10 µL and 20 µL on a 1–2% agarose gel with a 100 bp DNA marker. Loading different amounts helps to avoid under- or overloading
  8. Observe which of the shearing conditions gives a smear of DNA in the range of 200–1000 bp.
  9. Repeat optimization of the shearing conditions if the results indicate that the resulting DNA is not in the desired size range. Once optimal conditions have been determined, it is advised that you do not alter the cell concentration or volume of lysate per microcentrifuge tube for subsequent chromatin immunoprecipitation experiments.

DNA Purification

(The following protocol was adopted from Sambrook, et al., 2006.)

  1. Centrifuge your samples at full speed for 5 min at RT.
  2. Transfer supernatants to fresh microcentrifuge tubes.
  3. You can now purify your DNA using spin columns. For solvent extraction continue to Step 4.
  4. Transfer the nucleic acid sample to a polypropylene tube and add an equal volume of phenol:chloroform. The nucleic acid will tend to partition into the organic phase if the phenol has not been adequately equilibrated to a pH of 7.8-8.0.
  5. Mix the contents of the tube until an emulsion forms.
  6. Centrifuge the mixture at 80% of the maximum speed that the tubes can bear for 1 minute at room temperature. If the organic and aqueous phases are not well separated, centrifuge again for a longer time.
  7. Normally, the aqueous phase forms the upper phase. However, if the aqueous phase is dense because of salt (> 0.5 M) or sucrose (> 10%), it will form the lower phase. The organic phase is easily identifiable because of the yellow color contributed by the 8-hydroxyquinoline that is added to phenol during equilibration.
  8. Use a pipette to transfer the aqueous phase to a fresh tube. For small volumes (< 200 µL), use an automatic pipettor fitted with a disposable tip. Discard the interface and organic phase.
  9. Repeat Steps 1–4 until no protein is visible at the interface of the organic and aqueous phases.
  10. Add an equal volume of chloroform and repeat Steps 2–4.
  11. Recover the nucleic acid by standard precipitation with ethanol.

For more information about ChIP experiment guides, troubleshooting tips and supplementary protocols, please view our Chromatin Immunoprecipitation (ChIP) page.