NextSeq 500 paired end sequencing; Inhibition of the histone methyltrasferares DOT1L deregulates hormone responsive breast cancer cells methylome
Inhibition of the histone methyltrasferares DOT1L deregulates hormone responsive breast cancer cells methylome
Human luminal ERα-positive MCF-7 breast cancer cell line (ATCC, #HTB-22) was purchased from the American Type Culture Collection (ATCC). MCF-7 was cultured in Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich) supplemented with 10% FBS (HyClone, Milan, Italy) and 100 U/ml penicillin, 100 mg/mL streptomycin and 250 ng/mL Amphotericin-B. MCF-7 cells were routinely tested for Mycoplasma contamination with a PCR mycoplasma detection kit (Abm, Richmond, BC, Canada). Genomic DNA were extracted from three independent biological replicates of MCF-7 cells in exponentially growing condition, treated with 6.4 μM of selective DOT1L inhibitor, the EPZ004777 namely EPZ (S7353, Selleckchem), for 6 days or with 100 nM of selective estrogen receptor degrader, Fulvestrant (ICI182, 780) namely ICI. To this aim, cell pellets were resuspended in 2 volumes of lysis buffer (10 mM Tris EDTA pH 8.4, 100 mM NaCl) supplemented with 3% sodium dodecyl sulphate and Proteinase K (Thermo Fischer scientific) and incubated for 15 minutes at 60°C under gentle shaking. Then, NaCl saturated was added and centrifuged for 15 min at 25000 rpm at 4 °C. Supernatants were then collected, diluted in 2 volume of ethanol and centrifuged for 10 min at 25000 rpm at 4 °C. DNA pellets were washed, resuspended and quantified. Three independent biological replicates of MCF-7 cells following 6 or 3 days of treatment with vehicle (DMSO) were used as control performed in parallel. DNA purity was determined by using NanoDrop spectrophotometer ND-2000 (Thermo Scientific) through the evaluation of the absorbance ratio A260/A280. DNA concentration was determined by using Quant-IT DNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies) and its quality and integrity assessed with the Agilent 4200 Tapestation System (Agilent Technologies). Genomic DNA were extracted from three independent biological replicates of MCF-7 cells in exponentially growing condition, treated with 6.4 μM of selective DOT1L inhibitor, the EPZ004777 namely EPZ (S7353, Selleckchem), for 6 days or with 100 nM of selective estrogen receptor degrader, Fulvestrant (ICI182, 780) namely ICI. To this aim, cell pellets were resuspended in 2 volumes of lysis buffer (10 mM Tris EDTA pH 8.4, 100 mM NaCl) supplemented with 3% sodium dodecyl sulphate and Proteinase K (Thermo Fischer scientific) and incubated for 15 minutes at 60°C under gentle shaking. Then, NaCl saturated was added and centrifuged for 15 min at 25000 rpm at 4 °C. RRBS libraries were constructed according to the manufacturer's instructions. In detail, 2 μg of genomic DNA were used for each library preparation. DNA was digested with the MspI enzyme (CCGG site) at 37 °C for 16 h for each sample. The digested products were purified with the GeneJet PCR Purification Kit (Thermo Fisher Scientific) and libraries were prepared with TruSeq Library Prep Kit (Illumina). The recovered DNA was treated with the EZ DNA Methylation-Gold Kit (Zymo Research) for the bisulfite conversion. The converted DNA was amplified using PfuTurbo Cx Hotstart DNA Polymerase (Agilent Technologies).