NextSeq 500 sequencing; Histone ChIPSeq of H3K79me2 before and after EPZ004777 treatment in MCF7 breast cancer cells
Histone ChIPSeq of H3K79me2 before and after EPZ004777 treatment in MCF7 breast cancer cells
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 For histone extraction 100.000 cells/well were seeded in 35 mm plates were incubated with increasing concentrations of Dot1L inhibitors: EPZ004777 (S7353, Sellekchem), EPZ5676 (S7062 Sellekchem) and SGC0946 (S7079, Sellekchem) and their vehicle for 6 days. For this experiment we use only EPZ004777 (S7353, Sellekchem) at 6.4 microM. Cells were harvested, washed twice with ice-cold PBS and the pellets were resuspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v), 2 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed on ice for 10 min and nuclear separation was obtained by centrifugation at 6,500 x g for 10 min at 4°C. Then, the nuclei were resuspended in 0.2 N HCl before acid extraction of histones over night at 4°C. The histone content was determined using the Bradford assay and was analyzed by Western blot procedure. Antibody used:: Rb pAb Histone3 (di methyl k79); catalogue no: ab3594; Company: abcam After binding, beads washing, elution, reverse crosslinking and DNA extraction were performed as described in Tarallo et al, 2014. Three biological replicates were performed for each experimental condition. Then, 1µl aliquot of obtained DNA was used 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
EPZ004777
Experimental Factor: dose
6.4
Experimental Factor: immunoprecipitate
anti-H3K79me2