Nuclei preparation
Up to 3 × 107 cells were washed in ice-cold PBS and resuspended in 2 ml of ice-cold cell lysis buffer [300 mM sucrose, 10 mM Tris pH 7.4, 15 mM NaCl, 5 mM MgCl, 0.1 mM EGTA, 60 mM KCl, 0.2% NP-40, 0.5 mM DTT, 0.5 μM spermidine, 1× protease inhibitor (complete, Roche)]. After 5 min, the lysed cells were spun at 500 g for 5 min at 4°C with brakes off. After careful removal of supernatant, the nuclei were gently resuspended in 200 μl of ice-cold reaction buffer (20 μl 10× DNaseI buffer, 4 μl glycerol, 176 μl water) using pipette tips with cut off ends. The nuclei were spun again at 500 g for 5 min at 4°C and, following supernatant removal, were resuspended in 30 μl of reaction buffer per DNaseI condition. For example, if 2 × 107 cells were taken to look at four different conditions (e.g. 0, 20, 60, 120 units DNaseI), the nuclei were resuspended in 120 μl of reaction buffer. Separate 30 μl aliquots were then taken and gently mixed with 70 μl of DNaseI mix (see Table 1) on ice. This made a final digestion volume of 100 μl for each sample, which was left to incubate for 1 h on ice in the cold room.
Table 1. DNaseI mixes of 70 μl aliquots were prepared as detailed The resuspended nuclei were added as 30 μl aliquots to make a final digestion volume of 100 μl.
After 1 h, 700 μl of nuclei lysis buffer (100 mM tris HCL pH 8, 5 mM EDTA pH 8, 200 mM NaCl, 0.2% SDS) was added to each sample with 50 μg proteinase K. Following gentle mixing with inversion, the lysed samples were incubated at 55°C for 1 h. RNaseA (10 μg (Ambion)) was then added to each sample and further incubated at 37°C for 30 min. DNA was then extracted using standard phenol–chloroform techniques. Care was taken to use cut-off tips and very gentle pipetting to reduce non-specific DNA sheering. Following precipitation, DNA was resuspended in 200 μl of 0.1 TE and quantified using spectophotometry. Samples (1 μg) were analysed using gel-electrophoresis, as shown in Figure 1A.