Formaldehyde Crosslinking and DNA Fragmentation

1. Prepare crosslinking solution by diluting 35% formaldehyde in formaldehyde dilution buffer to reach a final concentration of 11%. Add crosslinking solution to the freshly isolated islets maintained in either culture medium (generally RPMI supplemented with 10% fetal calf serum) or Hank's balanced salt solution (HBSS) to reach a final concentration of 1% formaldehyde (see Note 1). Incubate 10 min at room temperature (see Note 2).

2. Stop fixation by adding 1.25 M glycine to attain a final concentration of 125 mM. Glycine quenches the fixation reaction by providing excess amino groups.

3. Centrifuge islets at 2000g and discard supernatants. Resuspend pellets in 1 mL phosphate-buffered saline (PBS) buffer and transfer to an Eppendorf tube. Wash the cells twice in PBS, centrifuging each time, to remove formaldehyde remains. At this point, the addition of protease inhibitors to the buffers or processing at low temperature is not necessary as proteins are fixed.

4. Centrifuge samples in microfuge at maximal speed for 2 min. Resuspend pellets in 0.2-0.4 mL SDS lysis buffer at room temperature (see Note 3). Incubate 10 min at room temperature with gentle agitation (e.g., in a rotating wheel). Incubation with SDS will break the cellular and nuclear membranes and expose the fixed chro-matin, thus facilitating sonication.

5. Sonicate samples to generate ideally fragments with an average size of 0.4-2 kb (see Note 4). For a volume of 150-400 iL in an Eppendorf tube, we insert the soni-fier tip to a distance of about 5-10 mm from the bottom of the tube, without touching the walls (see Note 4). Sonicate at maximum power for six 30-s intervals at continuous setting, maintaining samples on ice to minimize foaming. Foaming occurs when air is aspirated into the sample by turbulences. As the sample is in a high concentration of SDS, foaming will inevitably occur. Wait several minutes between pulses to allow for foaming to subside. Fragment size should be checked by gel electrophoresis in a parallel experiment or in an aliquot of the sample by reverting the crosslinking and extracting DNA, as described below. When optimizing conditions for a new antibody, it is usually a good idea to store samples at 4°C while crosslinking is reverted in an aliquot and the DNA size is checked, so that the immunoprecipitation experiment may be continued only if sonication is adequate. Fixed chromatin samples can be stored at 4°C for at least 1 wk if protease inhibitors are added. Figure 2 shows adequately sized DNA fragments obtained from 100 islets in a 150-iL volume.

6. After sonication, centrifuge samples in microfuge at maximum speed (20,000g) for 1 h at 4°C to remove cellular debris and high-molecular-weight DNA-protein aggregates.

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