CUT&RUN LoV-U for SMAD4 in human melanoma cells
CUT&RUN LoV-U for SMAD4 in human melanoma cells
Cells were detached with 2 mM EDTA-PBS for 5 min and then centrifuged 250 g for 5 min. Cells were resuspended in 2 ml PBS and counted; 250,000 cells were taken per replicate (N = 2 per condition). M170117 cells were cultured in at 37°C, 5% CO2, and 95% humidity in RPMI medium (11875093, Thermo Scientific) supplemented with 10% Calf Bovine Serum (12133C, Sigma-Aldrich), 10 U/ml Penicillin-Streptomycin (15140122, ThermoFisher Scientific) and 4mM L-Glutamine (25030, ThermoFisher Scientific). Passages were performed with 2 mM EDTA-PBS (AM9260G, ThermoFisher Scientific).  Stimulations were performed with TGFβ1 (RnD Systems 7754-BH-005, Stock: 10 µg/mL in 4mM HCl + 0.1% BSA, 1:5000 dilution of stock), and/or MEKi (Trametinib, Stock: 50 µM Active Biochemicals - A-1258, 1:1000 dilution of stock), and/or DMSO 20 hours prior to harvest for CUT&RUN. CUT&RUN Low Volume Urea was performed as described in (36355069), using anti-SMAD4 (ABIN6972727, antibodies-online), anti-SMAD4 (D3R4N #46535, Cell Signaling Technology), and IgG negative control (ABIN101961, antibodies-online). Library preparation was performed as described in (36355069) using the KAPA Hyper Prep Kit (Roche) and KAPA Duel Index Adapters.