Digital DNaseI mapping was performed essentially as described in28 (link). Briefly, 3134 and AtT-20 cells were
grown as described above. 1×108 cells were pelleted and
washed with cold phosphate-buffered saline. We resuspended cell pellets in
Buffer A (15 mM Tris-Cl (pH 8.0), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (pH 8.0), 0.5
mM EGTA (pH 8.0), 0.5 mM spermidine, 0.15 mM spermine) to a final concentration
of 2×106 cells/ml. Nuclei were obtained by dropwise addition
of an equal volume of Buffer A containing .04% NP-40 to the cells,
followed by incubation on ice for 10 min. Nuclei were centrifuged at
1,000g for 5 min, and then resuspended and washed with 25
ml of cold Buffer A. Nuclei were resuspended in 2 ml of Buffer A at a final
concentration of 1×107 nuclei/ml. We performed DNaseI (Roche,
10–80 U/ml) digests for 3 min at 37 °C in 2 ml volumes of DNase
I buffer (60 mM CaCl2, 750 mM NaCl). Reactions were terminated by
adding an equal volume (2 ml) of stop buffer (1 M Tris-Cl (pH 8.0), 5 M NaCl,
20% SDS, 0.5 M EDTA (pH 8.0), 10 μg/ml RNase A, Roche) and
incubated at 55 °C. After 15 min, we added Proteinase K (25
μg/ml final concentration) to each digest reaction and incubated them
overnight at 55 °C. After DNase I treatments, careful phenol-chloroform
extractions were performed. Control (untreated) samples were processed as above
except for the omission of DNase I. DNaseI double-cut fragments and sequencing
libraries constructed as described in 29 (link),30 (link).