Protein a g magnetic beads

ChIP assays were carried out as described26 (link)48 (link). In brief, MCF7 cells grown in medium supplemented with CD-FBS in T75 tissue culture plates for 72 h were treated without or with 10⁻⁹ M E2 for 1 h. Cells were then fixed with 0,75% paraformaldehyde at room temperature for 10 min and lysed with Nuclei Lysis Buffer containing 1% SDS and sonicated. Cell debris was pelleted and supernatant was collected. After blocking, the supernatant was incubated with a ChIP specific ERα antibody (HC-20x; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or IgG (Santa Cruz) for overnight and subjected to precipitation with Protein A/G Magnetic Beads (New England BioLabs). After de-crosslinking and protein digestion, DNA was recovered with a PCR Cleaning Kit (Qiagen). Samples (2 or 4 μl of a 30 μl elution) were subjected to PCR and RT-qPCR using primers specific for the region containing ERE of CXXC5. RT-qPCR results were normalized using percent (%) of input approach53 . For the RT-qPCR, CXXC5 ChIP primers (Forward Primer, FP: 5′-AATGCCTGGTCAAGCACATG-3′ and Reverse Primer, REP: 5′-TCTTCACTCTGTCACAAGAGGA-3′) or TFF1 ChIP primers (FP: 5′-CCTGTGGCCCAGCCACTGCGTCTTTCAG-3′ and REP: 5′-CCTATCTCCTTGGGAGAGCTGTGAG-3′) were used.