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Protocols used in ChIP-on-chip experiments

Cross-linking of cells and fragmentation and immunoprecipitation of chromatin:

Formaldehyde cross-linking and preparation of chromatin   10⁹ cells suspended in growth medium are transferred as 40ml aliquots to 50 ml tubes and placed on ice for 10 minutes. Then 1/10 volume of cross-linking solution is added directly to each tube. After a 10 minut e incubation on ice (adjust cross-linking time and temperature depending upon the cell type), 1/20 volume of 2.5M glycine solution is added to each tube to stop the cross-linking reaction. The cells are harvested by centrifugation at 2000 x g for 10 minut es at 4ºC. The cell pellets are re-suspended in cold PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) and washed twice . The final cell pellet may be snap-frozen in liquid nitrogen. Adherent cells can also be fixed directly on plates, treated with glycine, washed with PBS, and harvested in cold PBS with a silicone scraper.

The cell pellet is re-suspended in 30 ml of chilled Lysis Buffer 1 by pipetting and is mixed for 10 minutes at 4°C on a rocking platform. After centrifugation at 2000 x g for 10 minutes at 4°C, the cell pellet is re-suspended in 24 ml Lysis Buffer 2 by pipetting and mixed gently at room temperature for 10 min on a rocking platform. After centrifugation at 2000 x g for 10 minutes at 4°C, the pellet is resuspended in 5 ml of Lysis Buffer 3.

The mixture is divided into 5 ml aliquots in 15 ml conical tubes. These tubes are then placed into 50ml tubes containing ice. A sonicator (Branson Sonifier 450, with power setting at 5) is used to disrupt the cell and nuclear membranes and fragment the chromatin. To avoid foaming, the sonicator probe tip is first immersed in the mixture followed by a 25~30 second, continuous pulse of sonication. The tube is immediately placed on ice for at least 1 minute to avoid over-heating the sample. Sonication i s repeated until the chromatin fragments are of the desired length. The fragment size can be examined by agarose gel electrophoresis of 10 µl of cell extract digested with Proteinase K for 1 hour. The number of sonication cycles varies with cell type an d cross-linking conditions, and pilot tests are recommended. Ten cycles of sonication are normally sufficient to achieve the desired (~500 bp) fragment size. Finally, the chromatin solution is adjusted to 0.5% Sarkosyl (sodium lauryl sarcosine) and gentl y mixed for 10 minutes at room temperature on a rocking platform. The chromatin solution is then transferred to a centrifuge tube and spun for 10 min at 10,000 x g to remove cell debris. The supernatant is collected for chromatin immunoprecipitation. At this stage, the concentration of DNA should be normalized to 2mg/ml plus 10% glycerol. The solution can be stored at -80°C in 1 ml aliquots. The amount of chromatin generated here should be sufficient for 10 immunoprecipitation reactions.

Immunoprecipitation of chromatin   Magnetic beads (Dynal) pre-bound to polyclonal antibodies are used to immunoprecipitate the DNA associated with the protein of interest. To prepare the magnetic beads, 100 µl of sheep anti-rabbit IgG-conjugated Dynabeads (Dynal) are first wash ed three times with cold PBS containing 5 mg/ml Bovine Serum Albumin (BSA) and then resuspended in 5 ml of cold PBS. 10 µg of rabbit polyclonal antibody is added to the mixture and incubated overnight on a rotating platform at 4°C. After collecting the ma gnetic beads by centrifugation and washing three times with cold PBS containing 5 mg/ml BSA, the beads are re-suspended in 100 µl of cold PBS with 5 mg/ml BSA and are then ready for immunoprecipitation.

In an Eppendorf tube, 2 mg of soluble chromatin are first adjusted to 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitor cocktail and 1X TE to make 1.3ml total volume, then mixed with 100 µl of magnetic beads pre-bound to the antibody. The m ixture is incubated at 4°C overnight on a rotating platform. The magnetic beads are then collected using a magnet (Dynal), and the supernatant is removed by aspiration. To remove material non-specifically bound to the beads, 1ml of RIPA buffer is added t o the tube, and the beads are gently re-suspended by removing magnet and inverting by hand. The magnetic beads are again collected with the magnet and washed with RIPA buffer a total of 8 times. After washing once with 1 ml of TE, the beads are precipitated with magnet and the suspension is removed. The beads are then collected by centrifugati on at 2000 x g for 3 minutes and re-suspended in 50 µl of elution buffer. To elute precipitated chromatin from the beads, the tubes are incubated at 65°C for 10 minutes with constant agitation then centrifuged for 30 seconds at maximum speed in microcentr ifuge (14000rpm). 50 µl of supernatant are removed and mixed with 120µl of TE with 1% SDS. This solution is incubated at 65°C overnight to reverse the cross-links. As a chromatin input control, 50 µg of chromatin are mixed with 120 µl of TE containing 1% SDS in a separate tube and incubated at 65°C overnight.

Purification of DNA   After reversal of cross-links, proteins in the DNA sample are removed by incubation with 150 µl of Proteinase K solution (2% glycogen, 5% Proteinase K stock solution, and TE) for 2 hours at 37°C. The sample is then extracted twice with phenol and once with 24:1 chloroform/isoamyl alcohol. The sample is adjusted to 200 mM NaCl. After ethanol precipitation, the DNA is dissolved in 30 µl of TE containing 10 µg of DNase-free RNase A and incubated for 2 hours at 37°C. The DNA at this step can be f urther purified with a Qiaquick PCR clean-up kit (Qiagen).