MCF7 mock, FN-treated and recultured cells were used in this assay. After the cultures, the medium was aspirated and 1% formaldehyde in 10 mL of PBS was added. After 10 min of incubation at 37°C, the reaction was stopped with 2.5 M glycine. Approximately 2.5 ml of scraping buffer (PBS containing 1x protease inhibitor cocktail) was added to the plate, and cells were scraped from the plastic surface. The cells were centrifuged at 800×g at 4°C for 10 min and the pellet cells were lysed by lysis buffer supplied by MAGnify ChIP System (Invitrogen). The lysates were sonicated with Covaris sonicator for 13 cycles of 60 sec (under the conditions Duty - 5% Intensity - 2 Cycles/Burst - 200) and centrifuged at 14,000×g for 10 min at 4°C. Then, the supernatant was divided into 4 aliquots and immunoprecipitated with H3K27me3, H3K4me3, H3 and IgG (negative control) antibodies at a concentration of 2 µg/ml. The following steps were realized according to the MAGnify ChIP System (Invitrogen) manufacture’s instructions. The samples were quantified using Nanodrop 2000 and subjected to qPCR analysis.
For the ChIP qPCR assay, the following controls were tested at 400 nM: MYOD1 and GAPDH (Qiagen). To compare all the samples, the endogenous GAPDH gene was chosen. We used 25 ng of immunoprecipitated DNA in qPCR with SYBR Green (Applied Biosystems) mix. B1 primers were used as described [12] (link).
For the ChIP qPCR assay, the following controls were tested at 400 nM: MYOD1 and GAPDH (Qiagen). To compare all the samples, the endogenous GAPDH gene was chosen. We used 25 ng of immunoprecipitated DNA in qPCR with SYBR Green (Applied Biosystems) mix. B1 primers were used as described [12] (link).