Illumina HiSeq 4000 sequencing; ChIP-seq experiments of Estrogen Receptor Alpha (ERa) in the human breast cancer cell line T47D stably expressing GFP or neoGATA3
ChIP-seq experiments of Estrogen Receptor Alpha (ERa) in the human breast cancer cell line T47D stably expressing GFP or neoGATA3
T47D cells were grown until 70-80% confluence, treated 24h with 10nM E2, and cross-linked in 1% formaldehyde at room temperature for 10 min. After quenching, cells were harvested in PBS, nuclei were enriched with hypotonic solution, and lysed with 0.5% SDS. Chromatin was sonicated for 15 min in a Bioruptor water bath (Diagenode) using high intensity and 30”on/30”off cycles and then diluted to adjust SDS concentration to 0.1%. Immunoprecipitation was performed overnight at 4 °C with protein A/G agarose beads linked to anti-ERα (sc-543X) or rabbit IgG. Beads were washed 6x after incubation and the complex was eluted and de-crosslinked by overnight incubation at 65 °C. Eluted DNA was treated with proteinase K and purified with phenol-chloroform followed by isopropanol precipitation. Samples were diluted and pooled into NGS libraries. The E7645S NEBNext Ultra II DNA Library Prep Kit for Illumina and the E7335S NEBNext Multiplex Oligos for Illumina (Index Primer Set 1) were used according to the manufacturer's recommendations. Library concentrations were quantified with the Qubit Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using the Bioanalyzer (Agilent).