ChIP-seq Protocol for Transcription Factor Binding

The ChIP assay was performed according to the protocol from the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) as described in detail in Vydra et al., 2019 (link). For each IP reaction, 30 µg of chromatin and 4 μl of mouse anti-ERalpha monoclonal antibody (C15100066, Diagenode) was used. For negative controls, chromatin samples were processed without antibody (mock-IP). Obtained DNA fragments were used for global profiling of chromatin-binding sites or gene-specific ChIP-qPCR analysis using specific primers covering the known EREs. The set of delta-Cq replicates (difference of Cq values for each ChIP-ed sample and corresponding input DNA) for control and test sample were used for ERα-binding calculation (as a percent of input DNA) and estimation of the p-values. ERE motifs in individual peaks were identified using MAST software from the MEME Suite package (v. 5.1.1) (Bailey et al., 2015 (link)). The sequences of used primers are presented in Supplementary file 7.