Peo4 cells

To confirm predicted binding sites of the regulators FOXA1, PITX1, and ERα, we used PEO4 cells, derived from a poorly differentiated serous ovarian adenocarcinoma. PEO4 cells were obtained from Sigma-Aldrich (St. Louis, MO). Cells were grown to confluency in T75 flasks and crosslinked with 1/10th the volume of formaldehyde solution (Imquest Bio, Fredrick, MD). Flasks were agitated for 15 min at room temperature to fix the cells. Fixation was stopped by adding 1/20th volume of glycine solution to the existing media in the flask. The cultures sat at room temperature for 5 min. Following the incubation, cells were scraped from the flask, combined, and enumerated using trypan blue exclusion methodology. Two million cells were distributed in two conical tubes and pelleted by centrifugation at 800 x g at 4 °C for 10 min. The supernatant was removed, and the cells were resuspended in 10 mL chilled PBS-Igepal. The cells were pelleted a second time. The supernatant was removed, and the pellet resuspended in 10 mL PBS-Igepal. Next, 100 mL 1 mM phenylmethanesulfonyl fluoride was used to resuspend the pellet. The cells were centrifuged a third time, and the supernatant was removed entirely from the cell pellet. The cell pellets were snap-frozen on dry ice and stored at − 80 °C.