T47D WT and Y537S ESR1 cells (shGFP and shPR) were hormone-starved for 48h and then treated with vehicle or R5020 (10 nM, 1h). 1 x 10e6 cells were harvested in ice-cold PBS. Cells were crosslinked in 1% formaldehyde in PBS. Crosslinking was quenched by adding glycine at a final concentration of 125 mM. Crosslinked cell pellets were snap-frozen and stored at -80°C. For each ChIP experimental replicate, 2 x 10e6 crosslinked cells (from 2 crosslinked aliquots) were lysed in lysis buffer with PICS III using sonication (high, 30 seconds on/off, for 5 intervals of 10 minutes). 5% of lysate was reserved for input control and snap frozen to store at -80°C. Lysates were diluted to 1 µg/µl protein based on Nanodrop A280 concentrations and divided into 1 ml aliquots. 5 µg of the appropriate antibodies (phospho-PR [S294] for phospho-PR ChIP, rabbit IgG for phospho-PR negative control) were added to the appropriate lysate aliquots and rotated at 4°C overnight. Protein-chromatin was isolated and eluted using protein G beads (Cytiva Streptavidin Mag Sepharose Magnetic Beads, Thermo Fisher). Eluted ChIP samples were incubated with RNAse A and Proteinase K to reverse the crosslinked protein-chromatin. Input samples and ChIP-DNA were purified using a Qiagen QIAquick PCR Purification Kit, and purified DNA samples were eluted in 30 µl nuclease-free water. ChIP-DNA was quantitated by Qubit (Life Technologies), libraries were generated using the Illumina ChIP-SEQ kit and quality assessed using Bioanalyzer (Agilent). Sequencing was performed on the Illumina NovaSeq X by the University of Chicago Functional Genomics core. Samples were analyzed over 50M clusters per sample, which was variable due to the available library content.