While breast cancer mortality declines, hormonally driven breast cancer incidence continues to rise. Apart from a surgical mastectomy, the only preventative is systemic estrogen blocking, which has many side effects that limit uptake and adherence. Understanding the mechanisms driving increased incidence can help develop targeted preventatives to boost uptake and reduce cases. Our lab's preliminary data suggest estrogen receptor-positive (ER+) luminal progenitors (LPs) in the normal breast are the origin of ER+ breast cancer. Under protective conditions, ER+LPs decrease and change transcriptionally, with FOXA1 activity dramatically reducing. FOXA1, a key pioneer factor, has been studied in ER+ breast cancer, highlighting its integral role in estrogen signalling. However, its binding in normal breast tissue under high-risk conditions, which could influence tumorigenesis, remains unexplored, which could help develop cell- specific preventative treatments. We optimised a protocol (CUT&RUN) to profile FOXA1-DNA binding in ER+LPs, which we can only obtain in low cell numbers. We aimed to determine if 100,000 MCF7 cancer cells could be used to evaluate FOXA1- DNA binding and whether this could also work in normal breast cells. We found that 100,000 MCF7 cells were unsuitable for assessing FOXA1-DNA interactions due to the absence of specific peaks, and a high background. We therefore increased our cell input to 350,000 for ER+LP CUT&RUN sequencing, which returned excellent data quality with limited background noise. For future human studies on FOXA1 binding in high and low-risk states, it was concluded that these were the optimal cell input and sequencing parameters.