PRO-seq assay in human breast cancer MCF7 cells

PRO-seq assay was performed to detect the function of Top1 in the activation of acutely activated phase-separated MegaTrans enhancers. 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). 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. For knock-down of Top1 genes, the siTop1_1 and siTop1_3 were employed.