ChIP-seq Analysis of ER and FoxA1 in Breast Cancer

MCF-7, ZR75-1, T-47D and BT-474 human cell lines were obtained from ATCC and grown in the relevant media. TAM-R cells^{13 (link)} were a kind gift from Dr Iain Hutcheson and Prof. Robert Nicholson (Cardiff). The ER+ breast cancer tumours were obtained from the Nottingham Tenovus primary breast cancer series, Addenbrooke’s Hospital and Imperial College Healthcare NHS Trust, London, UK with appropriate ethical approval from the repositories. The malignant pericardial effusion and the two distant metastases were obtained from Imperial College Healthcare NHS Trust, London, UK. For ChIP in the tumours and metastases, the frozen sample was cut into smaller pieces prior to ChIP, which was then performed as previously described¹⁶ . For the malignant pericardial effusion, epithelial cells were first enriched using Dynabeads conjugated with Epcam^{17 (link)}. For ChIPs from cell line material, proliferating cells were cross-linked and processed for ChIP as previously described¹⁶ . The antibodies used were anti-ER (sc-543) from Santa Cruz Biotechnologies and anti-FoxA1 (ab5089) from Abcam. Sequences generated by the Illumina Genome Analyzer were processed by the Illumina analysis pipeline version 1.6.1, and aligned to the Human Reference Genome (assembly hg18, NCBI Build 36.1, March 2008) using BWA version 0.5.5¹⁸ . Differential binding analysis was performed using the DiffBind package¹⁹ . For immunohistochemical analyses, ER staining was conducted using the 6F11/2 mouse monoclonal antibody (Novocastra, Leica Microsystems, Bucks, UK) and FoxA1 staining was conducted using a rabbit polyclonal antibody (ab23738) from Abcam. An Allred scoring system was used to assess staining accounting for both staining intensity and the proportion of cells stained.