Chromatin Immunoprecipitation

1. Crosslink protein to DNA by adding 10 |L of 37% formaldehyde (1% final, see Note 2) directly to the nuclei extract and incubate on ice for 10 min and then at room temperature for 20 min.

2. Spin for 5 min at 6000g. Remove the supernatant and resuspend the pellet in 200 |L of lysis buffer. Incubate the mixture on ice for 10 min.

3. Sonicate the lysate to reduce DNA length to between 200 and 1000 bp (see Note 10). Keep the samples on ice all the time.

4. Remove debris by centrifugation for 10 min at 15,000g at 4°C.

5. Quantify the amount of DNA in the supernatant by measuring absorption at 260 nm. Dilute each sample to 0.1 |g/ |L in lysis buffer.

6. Take 200 |L of the diluted supernatant and dilute it 10-fold in ChIP dilution buffer.

7. Save 1% of this chromatin solution as the input control.

8. To reduce nonspecific background, preclear the chromatin solution with 80 |L of salmon sperm DNA/protein A agarose slurry for 30 min at 4°C with agitation.

9. Pellet beads by centrifugation for 3 min at 1000g at 4°C and collect supernatant.

10. Add 5 to 8 |L of an antibody (against the protein of interest) to 1 mL of the chromatin solution and incubate overnight at 4°C with rotation (10 rpm). The remaining 1 mL of the chromatin solution will be used as no-antibody control.

11. Precipitate the antibody-protein complexes by adding 60 |L of salmon sperm DNA/Protein A Agarose slurry and mixing by rotation at 4°C.

12. At this step, it is time to prepare elution buffer.

13. Pellet immunoprecipitate (agarose beads) by centrifugation (3 min at 1000g at 4°C).

14. Wash the beads for 5 min with rotation using 1 mL of each of the buffer listed below:

Low salt immune complex wash buffer.

High salt immune complex wash buffer.

LiCl immune complex wash buffer, and twice with TE.

15. Add 250 |L elution buffer to the pelleted beads. Vortex mix briefly and incubate at room temperature for 15 min with rotation. Centrifuge for 3 min at 1000g at room temperature and carefully transfer the supernatant to a new tube. Repeat the entire elution procedure and combine the eluates.

16. After the addition of 20 ||L of 5 M NaCl, reverse the crosslinks by incubating at 65°C for 4 h. Also, do not forget to do the same for the input control sample.

17. Add 10 |L of 0.5 MEDTA, 20 |L of 1 M Tris-HCl, pH 6.5, and 2 |L of 10 mg/mL proteinase K and incubate for 1 h at 45°C to degrade proteins.

18. The solution is extracted with phenol-chloroform and precipitated with ethanol to recover DNA (see Note 11). Wash pellets with 70% ethanol and dry it in a speedvac.

19. Resuspend the DNA pellet in 20 |L of water.

20. Detect specific sequences from no-antibody control, immunoprecipitated, input control, and unbound DNA samples by PCR. Conditions for PCR must be determined for each gene of interest. The PCR product size should be between

200 and 400 bp. Addition of labeled 32p-dCTP (1 |Ci) allows one to detect the products with high sensitivity by autoradiography. 21. Add 2 |L of 10X DNA loading buffer to 10 |L of the PCR product and load on a 6% non denaturing polyacrylamide Tris-borate EDTA (TBE) gel. After electro-phoresis, dry the gel before autoradiography (Ethidium bromide staining can be used as a semiquantitative assay if no radioactive label is used in the PCR).

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