GSM6387134: K562_BRD4_B1_t4; Homo sapiens; OTHER
For Chem-map experiment, U2OS cells were detached using accutase (Stem Cell Technologies, 07920) and quenched using complete culture media. U2OS and K562 cells were collected by centrifugation and fixed in 0.1% formaldehyde (Thermo Fisher Scientific, 28906) in PBS for 2 min at room temperature, followed by quenching with glycine (Sigma-Aldrich, 50046) to a final concentration of 75 mM. Fixed cells were collected by centrifugation at 600x g for 4 min, followed by resuspension in cold wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl and 0.5 mM spermidine (Sigma-Aldrich, S0266)) in nuclease-free water supplemented with a Complete Protease Inhibitor, EDTA-free (Sigma-Aldrich, 11873580001). [Note: For Chem-map of G4 ligands, NaCl was replaced by equivalent concentrations of KCl in all buffers to maintain G4 stability]. For 5 samples, 50 μL (for JQ1-btn) or 75 µL (for G4 ligands and biotinylated doxorubicin) concanavalin A beads (Bangs Labs, BP531) were washed twice in 1 mL binding buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2 in nuclease-free water) and resuspended in 75 µL binding buffer. 100 µL of cell suspension were incubated with 10 µL pre-washed concanavalin A beads at 25 °C for 10 min at 600 rpm. Beads-bound cells were gently washed twice with wash buffer, before resuspending in 100 µL probe-1st Ab pre-complex (small molecule concentration is 400 nM with a ratio of 1.2:1 to anti-biotin (D5A7) Rabbit mAb (Cell Signaling Technology, 5597)) in antibody buffer (2 mM EDTA, 0.1% BSA (Sigma-Aldrich, A8577), 0.05% digitonin (EMD Millipore, 300410) in wash buffer) and incubating at 4 °C overnight at 600 rpm. [Note: For Dox-btn1, final Dox-btn1 concentration is diluted to 0.2 µM]. Cells were washed 3x with 500 µL Dig-wash buffer (0.05% digitonin in wash buffer) and resuspended in 100 µL 2nd Ab-Tn5 transposome complex (2nd Ab (Antibodies-Online, ABIN101961, ~ 8 µM) ratio is 1:100; 2nd Ab and pA-Tn5 at a ratio of 2:1) in Dig-300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 0.01% digitonin in nuclease-free water supplemented with Complete Protease Inhibitor, EDTA-free) and incubated at 25 °