Protocol 141 Performing a successful ChIP assay

We have obtained the best results by using the following protocol, which is based on that described by Upstate Group, Inc and Spencer et al. (18):

1. Stimulate or treat cells as appropriate. Cells should be treated under conditions for which transcriptional activation of the gene of interest has been demonstrated. Use 1 x 106 cells for each antibody.

2. Crosslink histones and other nuclear factors to DNA by adding formaldehyde directly to culture medium to a final concentration of 1%. Incubate for 15-60 min (as previously determined) at room temperature. Preliminary experiments to estimate the best combination of crosslinking time and fragmentation should be performed. When planning to store crosslinked cells, glycine should be added to a final concentration of 0.125 M and incubated for 5 min. After that, 1 x phosphate-buffered saline (PBS) washes should be performed as described below.

3. Aspirate medium, removing as much of it as possible. Wash cells twice using ice-cold 1 x PBS containing protease inhibitors (there are several cocktails of protease inhibitors commercially available that cover a wide spectrum of inhibition).

4. Scrape cells and transfer to a conical tube. For suspension cells, the PBS washes need to be performed in the tube.

5. Pellet cells for 4 min at 2000 g at 4°C. Warm cell lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) to room temperature to dissolve precipitated SDS and add protease inhibitors.

6. Resuspend cell pellet in cell lysis buffer and incubate for 10 min on ice. Each 1 x 106 cells should be resuspended in 200 |l of cell lysis buffer.

7. Sonicate the cell lysate to shear DNA to lengths between 200 and 1000 bp, being sure to keep samples ice-cold. When optimizing sonication conditions, at this point, 20 |l of 5 M NaCl are added to each 500 | l and incubation at 65°C for 4 h is performed to reverse crosslinks. This incubation is followed by phenol/chloroform extraction and samples are analyzed in agarose gels to visualize shearing efficiency.

8. Once sonication conditions have been optimized, centrifuge samples following sonication for 10 min at 13 000 g at 4°C. The size of a sample will be the equivalent amount of 200 ^l of the sonicated cell lysate.

9. Dilute the sonicated cell lysate 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl), adding protease inhibitors, as above. This is done by adding 1800 ^l of ChIP dilution buffer to each 200 ^l of sonicated cell lysate to give a final volume of 2 ml for each immunoprecipitation condition. If proceeding to PCR a portion of the diluted cell pellet suspension, a small volume can be kept to quantitate the amount of DNA present in different samples for the PCR protocol. This sample is considered to be the input/starting material, and needs to be heated at 65°C for 4 h in order to reverse crosslinks.

10. To reduce nonspecific background, it is advisable to treat the diluted cell lysate with 80 ^l of protein A/G-agarose/salmon sperm DNA (50% gel slurry in TE buffer, containing 15 ^g of sonicated salmon sperm DNA; Upstate Group, VA) for 30 min at 4°C with agitation.

11. Pellet agarose beads by brief centrifugation and collect the supernatant fraction.

12. Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2 ml of supernatant fraction and incubate overnight at 4°C with rotation. For a negative control, perform a no-antibody immunoprecipitation and a pre-immune serum precipitation (when available).

1 3. Add 60 ^l of protein A/G-agarose/salmon sperm DNA beads for 1 h at 4°C with rotation to collect the antibody-protein complex.

14. Pellet agarose by gentle centrifugation (2000 g at 4°C for 1 min). Carefully remove the supernatant and keep this fraction, which consists of unbound DNA. Wash the protein A/G agarose beads for 5 min on a rotating wheel with 1 ml of each of the following solutions:

• Low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl)

• High-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl)

• LiCl wash buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1)


15. After the last washing, protein-DNA complexes can be eluted from the antibody by adding 250 |l of freshly made elution buffer (1% SDS, 0.1 M NaHCO3) to the pelleted protein A agarose-antibody-protein-DNA complex. Vortex briefly to mix and incubate at room temperature for 15 min with rotation. Pellet the agarose beads, and carefully transfer the supernatant fraction (eluate) to another tube and repeat elution. Combine eluates. The total volume should be approximately 500 |l.

1 6. Add 20 |l of 5 M NaCl to the combined eluates and reverse protein-DNA crosslinks by heating at 65°C for 4 h. The input sample as well as the unbound samples should also be treated in a similar manner by adding the equivalent volume of 5 M NaCl.

1 7. Add 10 |l of 0.5 M EDTA, 20 |l of 1 M Tris-HCl, pH 6.5 and 2 |l of 10 mg/ml proteinase K to the combined eluates and incubate for 1 h at 45°C.

18. After protein removal, add an equal volume of phenol:chloroform:isoamyl alcohol (24:23:1) to the sample and mix, then centrifuge at 12 000 g for 1 min. The upper aqueous phase is transferred to a new microcentrifuge tube and phenol:chloroform extraction is repeated. The DNA is then precipitated by adding one-tenth of the volume of sodium acetate to the aqueous phase, a carrier, such as glycogen or yeast tRNA, and 2.5 volumes of absolute ethanol, and then incubating the sample at -80°C for at least 1 h. After that, wash the DNA pellet with 70% ethanol and air dry.

19. Resuspend each pellet in 20 |l of water for PCR analysis. In standard ChlPs, the input sample is used at different dilutions to establish conditions for which specific PCR products are obtained below saturation.

Turning photons into results: principles of fluorescent microarray scanning

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