| Construction Protocol | BC-3 cells were grown on RPMI 1640 medium supplemented with 15% FCS (Invitrogen), 100 U/ml penicillin G, and 100 g/ml streptomycin BC-3 cells were cross-linked with 1% formaldehyde in PBS at room temperature for 10min. After a wash in PBS containing 125 mM glycine, cells were collected by centrifugation and sonicated in lysis buffer containing 0,1% SDS, 0,1% sodium deoxycholate, 1 mM EDTA, 10 mM TrisHCl pH 8.0, 140 mM NaCl, 1% Triton X-100 and protease inhibitors to generate chromatin fragments of 100300 bp in length. Following a brief centrifugation (16.000xg, 20 min, 4 C), the fragmented chromatin was immunoprecipitated with a monoclonal antibody against p53 (Clone DO-1, GeneSpin) or control IgG (normal mouse IgG: sc-2025, Santa-Cruz Biotechnology). The precipitates were incubated at 65 C over night to reverse the formaldehyde crosslinking and samples incubated with proteinase K and RNase-A before phenol extraction and ethanol-precipitation of target DNA. Similarly as in Tuupanen et al. Nature Genetics 2009.PMID: 19561604 TPA was added to the cells for 4h or 24h with 20 ng/ml dose BC-3 cells were cross-linked with 1% formaldehyde in PBS at room temperature for 10min. After a wash in PBS containing 125 mM glycine, cells were collected by centrifugation and sonicated in lysis buffer containing 0,1% SDS, 0,1% sodium deoxycholate, 1 mM EDTA, 10 mM TrisHCl pH 8.0, 140 mM NaCl, 1% Triton X-100 and protease inhibitors to generate chromatin fragments of 100300 bp in length. Following a brief centrifugation (16.000xg, 20 min, 4 C), the fragmented chromatin was immunoprecipitated with a monoclonal antibody against p53 (Clone DO-1, GeneSpin) or control IgG (normal mouse IgG: sc-2025, Santa-Cruz Biotechnology). The precipitates were incubated at 65 C over night to reverse the formaldehyde crosslinking and samples incubated with proteinase K and RNase-A before phenol extraction and ethanol-precipitation of target DNA. IP-DNA fragments were first repaired using Klenow and T4 DNA polymerases and T4 polynucleotide kinase (MBI Fermentas, Latvia), and then ligated to adapters according to manufacturer's instructions (Illumina). PCR-amplified fragments of approximately 180-300bp were isolated. |
FTP
