Illumina HiSeq 2500 sequencing; ChIP-Seq of ERalpha and DOT1L in MCF7 cell before and after EPZ004777 and ICI 182,780 treatment
ChIP-Seq of ERalpha and DOT1L in MCF7 cell before and after EPZ004777 and ICI 182,780 treatment
ER-alpha positive BC cells: MCF-7 (ATCC HTB-22) were propagated in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS (HyClone) and antibiotics: 100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin-B. None ER-alpha positive BC cells: MCF-7 (ATCC HTB-22) were propagated in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS (HyClone) and antibiotics: 100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin-B.  ChIP-Seq experiments were performed in three biological replicates in MCF-7 cells, in exponential growing condition. Cells were treated with ICI 182,780 10-7 M for 3 days and with EPZ004777 6.4 µM or DMSO for 6 days.  ChIP-Seq experiments were performed in three biological replicates in MCF-7 cells, in exponential growing condition. Cells were treated with ICI 182,780 10-7 M for 3 days and with EPZ004777 6.4 µM or DMSO for 6 days.  We used antibodies to ERα (HC-20) (Santa Cruz Biotechnology, SC-8002), Dot1l Antibody (Bethyl Laboratories, A300-954A), and rabbit IgG antiobody as negative control. Cells lysis, sonication and binding were performed as described by Serandour AA et al, 2013. After binding, beads washing, elution, reverse crosslinking and DNA extraction were then performed as described in Ambrosino et al, 2010. 1µl aliquot of DNA obtained was analysed to asses size distribution by Agilent Genomic DNA ScreenTape & Reagents using an Agilent 4200 TapeStation System (Agilent Technologies). The concentration of each DNA sample was determined by using Quant-IT DNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies).   10 ng of each purified ChIP DNA were used for sequencing libraries preparation with TruSeq ChIP Sample Prep Kit (Illumina Inc.).
Experimental Factor: compound
fulvestrant
Experimental Factor: dose
100
Experimental Factor: immunoprecipitate
anti-Dot1l