GSM6387210: K562_Tucidino_DOX-btn1_200nM_B3_t5; 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 °C for 1 hr at 600 rpm. Cells were then washed 3x in 500 µL Dig-300 buffer before incubation in 300 µL tagmentation buffer (10 mM MgCl2 in Dig-300 buffer) at 37 °C for 1 hr at 600 rpm. After tagmentation, cells were washed twice with 500 µL TAPS wash buffer (10 mM [tris(hydroxymethyl) methylamino] propanesulfonic acid (TAPS, Alfa Aesar, J63268.AE), 0.2 mM EDTA in nuclease-free water). 150 µL of extraction buffer (0.5 mg/mL proteinase K (Thermo Fisher Scientific, EO0491), 0.5% SDS (Sigma-Aldrich, L4509) in 10 mM Tris-HCl, pH8.0) were added, vortexed and incubated at 55 °C for 1 hr at 800 rpm. Next, 150 µL phenol-chloroform-isoamyl alcohol (Invitrogen, 15593049) was added and mixed. The mixture was transferred to MaXtract High Density phase-lock tubes (QIAGEN, 129046) and centrifuged at room temperature for 3 min at 16,000x g. 150 µL chloroform were added to the top aqueous phase, mixed by inverting the phase-lock tubes for 10 times and centrifuged at room temperature for 3 min at 16,000x g. The top aqueous layer was transferred to a 1.5 mL DNA Lo-bind tube (Eppendorf, 022431021). 6 µL 5 M NaCl and 375 µL cold ethanol were added, mixed and incubated at -20 °C overnight. Samples were centrifuged at 4 °C for 30 min at 21,130x g. The supernatant was carefully poured off and the DNA pellet rinsed with 1 mL cold 100% ethanol followed by centrifugation at 4 °C for 2 min at 21,130x g. After pouring off the wash and draining the residual liquid with paper towel, the pellet was left to air-dry. Finally, the pellet was resuspended in 25 µL elution buffer 1 (10 mM Tris-HCl, pH8, 1 mM EDTA, 1/400 RNAse A (Thermo Fisher Scientific, EN0531) in nuclease-free water) by vortexing and incubating at 37 °C for 10 min at 800 rpm. For BRD4 CUT&Tag 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). ConA beads (Bangs Labs, BP531) per sample 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 50 µL anti-BRD4 (E2A7X) rabbit antibody (Cell Signalling Technology, 13440) in antibody buffer (2 mM EDTA, 0.1% BSA (Sigma-Aldrich, A8577), 0.05% digitonin (EMD Millipore, 300410) in wash buffer) at a ratio of 1:50 and incubating at 4 °C overnight at 600 rpm. Cells were washed 3x with 500 µL Dig-wash buffer (0.05% digitonin in wash buffer) and resuspended in 50 µL Guinea Pig anti-rabbit antibody (Antibodies-Online, ABIN101961) in Dig-wash buffer at a ratio of 1:100. Diluted pA-Tn5 adapter complex (1:250 ratio) was then added in Dig-300 buffer followed by the tagmentation reaction in tagmentation 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 °C for 1 hr at 600 rpm. Cells were then washed 3x in 500 µL Dig-300 buffer before incubation in 300 µL tagmentation buffer (10 mM MgCl2 in Dig-300 buffer) at 37 °C for 1 hr at 600 rpm. After tagmentation, cells were washed twice with 500 µL TAPS wash buffer (10 mM [tris(hydroxymethyl) methylamino] propanesulfonic acid (TAPS, Alfa Aesar, J63268.AE), 0.2 mM EDTA in nuclease-free water). 100 µL of extraction buffer (0.5 mg/mL proteinase K (Thermo Fisher Scientific, EO0491), 0.5% SDS (Sigma-Aldrich, L4509) in 10 mM Tris-HCl, pH8.0) were added, vortexed and incubated at 55 °C for 1 hr at 800 rpm. Next, 100 µL phenol-chloroform-isoamyl alcohol (Invitrogen, 15593049) was added and mixed. The mixture was transferred to MaXtract High Density phase-lock tubes (QIAGEN, 129046) and centrifuged at room temperature for 3 min at 16,000x g. 100 µL chloroform were added to the top aqueous phase, mixed by inverting the phase-lock tubes for 10 times and centrifuged at room temperature for 3 min at 16,000x g. The top aqueous layer was transferred to a 1.5 mL DNA Lo-bind tube (Eppendorf, 022431021). 4 µL 5 M NaCl and 250 µL cold ethanol were added, mixed and incubated at -20 °C overnight. Samples were centrifuged at 4 °C for 30 min at 21,130x g. The supernatant was carefully poured off and the DNA pellet rinsed with 1 mL cold 100% ethanol followed by centrifugation at 4 °C for 2 min at 21,130x g. After pouring off the wash and draining the residual liquid with paper towel, the pellet was left to air-dry. Finally, the pellet was resuspended in 25 µL elution buffer 1 (10 mM Tris-HCl, pH8, 1 mM EDTA, 1/400 RNAse A (Thermo Fisher Scientific, EN0531) in nuclease-free water) by vortexing and incubating at 37 °C for 10 min at 800 rpm. In a 0.2 mL PCR tube, 25 µL NEBNext® Ultra™ II Q5 2x PCR master mix (NEB, M0544), 2 µL of 10 µM uniquely barcoded v2 Ad1.x primer, 2 µL of 10 µM uniquely barcoded v2 Ad2.x primer and 21 µL tagmented DNA were added and subjected to PCR (72 °C for 5 min, 98 °C for 30 s, followed by 10 cycles of 98 °C for 10 s and 63 °C for 10 s, and one cycle of 72 °C for 1 min). Libraries were purified using 1.3x ratio (65 µL) Ampure XP beads (Beckman Coulter, A63882). After 10 min incubation at room temperature, bead-bound DNA was washed twice with 80% ethanol and libraries were eluted with 25 µL 10 mM Tris-HCl for 5 min at room temperature. [Note: PCR program condition for Dox-btn1 is 72 °C for 5 min, 98 °C for 2 min, followed by 10 cycles of 98 °C for 10 s and 63 °C for 10 s, and one cycle of 72 °C for 1 min, to maximally disrupt doxorubicin-DNA interaction]