title: EP300 genomic binding analysis in MCF7 cells with E-cadherin inhibition using ChIP-seq
design: EP300 genomic binding analysis in MCF7 cells with E-cadherin inhibition using ChIP-seq
library_construction_protocol: Cell Line: MCF7 (human breast cancer cell line, ATCC® HTB-22™)   Collection: MCF7 cells were cultured in six-well plates with pre-warmed Minimum Essential Medium Eagle (MEM), supplemented with 10% fetal calf serum (FCS). The cells were cultured until they reached 70-80% confluence. After treatment with either isotype control (cAb) or anti-E-cadherin neutralizing monoclonal antibody (nAb), cells were collected by scraping and washed twice with PBS.   Treatment: E-cadherin function was inhibited using 1 µg/mL of anti-E-cadherin antibody (SHE78-7) in culture media for 3 days. Control cells were treated with an equivalent concentration of isotype control antibody (cAb).   Preservation: Following treatment, cells were fixed with 1% formaldehyde for 10 minutes, followed by quenching with 0.125 M glycine. Cells were stored in PBS for further processing.  MCF7 cells were cultured in six-well plates (Greiner Bio-One, Kremsmünster, Austria) with 3 mL of pre-warmed Minimum Essential Medium Eagle (Sigma-Aldrich, MO, USA) supplemented with 10% (v/v) fetal calf serum (Life Technologies, CA, USA). The cells were maintained at 37°C with 5% CO2 to ensure proper growth conditions. The cells were passaged regularly, and for the experiment, they were allowed to reach 70-80% confluence before being treated with either neutralizing anti-E-cadherin antibody (nAb) or isotype control antibody (cAb) for 3 days. Following the treatment period, the cells were harvested for DNA and RNA collection.  MCF7 cells were treated with a neutralizing anti-E-cadherin monoclonal antibody (nAb) (mouse IgG; SHE78-7, Invitrogen, MA, USA) to inhibit E-cadherin function. The nAb was resuspended in nuclease-free water to a concentration of 1 µg/mL and added to the cell culture medium. The cells were treated daily for 3 days starting from day 1 after cell passaging. The control group was treated with an isotype control antibody (cAb) at the same concentration (1 µg/mL). Treatment was maintained for 3 days, after which the cells were harvested for subsequent analyses.     DNA Extraction and IP: Chromatin was extracted from MCF7 cells after fixation using formaldehyde, followed by chromatin shearing. For the immunoprecipitation, 30 µg of chromatin per IP was incubated with 5 µg of EP300 antibody (Santa Cruz, SC-585). An input control was also prepared for each antibody using an equivalent amount of chromatin to account for background, and the IP slurries were incubated overnight with the antibody at 4°C. The cell lysates were processed using a protease inhibitor cocktail, and the chromatin was purified using a PCR purification kit (QIAGEN, Hilden, Germany). The extracted DNA was then quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific).  DNA Purification: DNA was purified from the chromatin using a QIAGEN PCR Purification Kit, and the integrity of the DNA was verified by visualizing the samples on an agarose gel and by measuring absorbance at 260/280 nm using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).  Library Preparation: DNA libraries for ChIP-seq were constructed using the TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol. The extracted chromatin was fragmented into small pieces, and adapters were ligated to both ends of the fragments to prepare for sequencing.  Amplification and Quality Control: The library was amplified using PCR, and the quality was assessed using a Bioanalyzer (Agilent Technologies) to ensure that the fragment sizes were appropriate for sequencing. The concentration of the library was measured using a Qubit 3.0 Fluorometer.  