Supporting Materials and Methods Crosslinking of Cells, Fragmentation, and Immunoprecipitation of Chromatin

Formaldehyde Crosslinking. Cells (10) suspended in growth medium are transferred as 40-ml aliquots into 50-ml tubes and placed on ice for 10 min. Then 1/10 volume of crosslinking solution (11% formaldehyde/0.1M NaCl/1mM Na-EDTA/0.5 mM NaEGTA/50mM Hepes, pH 8.0) is added directly to each tube. After 10-min incubation on ice, 1/20 vol of a 2.5-M glycine solution is added to each tube to stop the crosslinking reaction. The cells are harvested by centrifugation at 2,000 × g for 10 min at 4oC. The cell pellets are resuspended in cold PBS (137 mM NaCl/2.7 mM KCl/10 mM Na2HPO4/2 mM KH2PO4) and washed twice. The final cell pellet is snap frozen in liquid nitrogen.