TOP1cc-seq assay in human cancer cells

TOP1cc-seq assay was performed for the quantitative detection of the transient DNA-protein covalent interactions upon acute stimulations. Overall design: MCF7 cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen). LNCAP cells were maintained at 37°C and 5% CO2 in Advanced RPMI 1640 medium (GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen). For hormone treatments, cells were incubated at 37°C and 5% CO2 for at least 3 days in phenol red-free DMEM (GIBCO/Invitrogen) supplemented with 5% charcoal dextran-stripped FBS (GIBCO/Invitrogen). 17-ß-Estradiol (E2; Steraloids, Inc.) was added to a final concentration of 100 nM. TNFalpha (R&D systems) was added to a final concentration of 20 ng/ml. 5a-dihydrotestosterone (DHT, Sigma) was added to a final concentration of 100 nM. The ethanol (EtOH) vehicle control was 0.05% in all samples.