Genome-Scale Biology Program, Research Programs Unit and Department of Pathology, Haartman Institute, University of Helsinki
INSDC center name
Genome-Scale Biology Program, Research Programs Unit and Department of Pathology, Haartman Institute, University of Helsinki
INSDC first public
2016-05-23T17:06:44Z
INSDC last update
2016-01-27T15:25:30Z
INSDC status
public
Submitter Id
E-MTAB-4356:Balistreri_p53_sample_Nutlin
broker name
ArrayExpress
cell line
BC-3
common name
human
compound
Nutlin-3
dose
10
sample name
E-MTAB-4356:Balistreri_p53_sample_Nutlin
time
8
Sequenced DNA Library
library_name
Balistreri_p53_sample_Nutlin
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_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 Nutlin 3 treatment for 8h with 10 M concentration. 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.