Formaldehyde cross-linking and cell lysis

Cells at mid-log phase were collected, resuspended in PBS, and then cross-linked with 0.01% formaldehyde at room temperature for 10 min with slow shaking. The cross-linking reaction was terminated by adding 1 M glycine-NaOH buffer (pH 8.0) to a final concentration of 0.125 M and additional shaking for 2 min. The cells were then washed once with ice-cold PBS buffer and the pellet was flash-frozen in liquid nitrogen, and stored at −80°C. Cell pellets were thawed by addition of ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, 175 mM KCl, 0.5% NP40, 1 mM DTT, and 120 U RNAse inhibitor) (New England Biolabs) supplemented with EDTA-free protease inhibitors (Roche). Samples were then subjected to three cycles of sonication (10 pulses of 0.5 s) followed by 1 min rest between cycles at 4°C (Branson Ultrasonics Sonifier S-250). The extract was then supplemented with 1x DNAse salt solution and 100 U DNAse I (Roche) and incubated for 30 min at 4°C on rotator. Samples were returned to ice and the reaction was immediately terminated by the addition of 10 mM EDTA and 10 mM EGTA. The extract was finally clarified by centrifugation for 20 min at 15,000 × g, the supernatant removed to a new microcentrifuge tube and protein concentration determined using the Bradford assay (Bio-Rad).