Chromatin immunoprecipitation (ChIP) assay and Real Time PCR (RT-PCR)

ChIP experiments were performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signalling Technology, Massachusetts, USA) according to the instructions provided by the manufacturer. Briefly, BON, CM and QGP1 cells were grown until 90% confluence was reached and DNA-protein crosslinking was achieved by adding 1% formaldehyde directly in the culture medium. Digestion and isolation of the nuclei was accomplished after incubating the samples with 5 ul of micrococcal nuclease for 20 minutes at 37 °C. The quality of the chromatin preparations was assessed by electrophoresis in an agarose gel and only samples showing a pattern of chromatin fragments ranging 100–1000 bps were used in the following steps: up to 5 ug of chromatin preparation was immunoprecipitated using 1 to 2 ug of the indicated antibodies and protein G magnetic beads. DNA was then eluted from the antibody/protein G beads, purified using spin columns and used as a template for PCR and RT-PCR experiments. PCR evaluation of eluted products was performed with the conditions described above. RT-PCR employed a Sybr Fast Master Mix (KAPA Biosystems Massachusetts, USA) with a program that consisted of 45 cycles of 30 seconds at 95 °C and 30 seconds at 62 °C. Aliquots of chromatin that were not immunoprecipitated (referred as “input”) were used to normalize the results, that was calculated using the following formula: 2^−(∆Ct), where Ct = Ct TERTp immunoprecipitated − Ct TERTp input.