Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
TP53

Cell type

Cell type Class
Bone
Cell type
Saos-2
Primary Tissue
Bone
Tissue Diagnosis
Osteosarcoma

Attributes by original data submitter

Sample

source_name
osteosarcoma cell line
cell line
SaOS2
chip antibody
monoclonal anti-p53 (Santa Cruz Biotechnology)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
The cells were harvested and washed once in 1% of the chilled phosphate-buffered saline (PBS). 1-5x107 cells were centrifuged at 500 x g for 5 min at 4oC then incubated in 1% formaldehyde for 15 min at room temperature and washed with chilled PBS. After a further centrifugation, cell pellets were resuspended in SDS lysis buffer (50 mM of Tris/HCl, pH 8.0, containing 1 mM of DTT, 1 mM of PMSF, 1 mM of Na3VO4, 10µg/ml of aprotinin, 1 µg/ml of leupeptin, 1 µg/ml of pepstatin A, 1% (w/v) of SDS and 10 mM of EDTA) and lysates were sonicated to obtain DNA fragments that consist of 200-500 bp in length. Immunoprecipitation was performed with mouse monoclonal anti-p53 (Santa Cruz Biotechnology) for 2 h at 4oC on the rotation. Immuno-complexes were collected with 30 µl of Protein G-Sepharose beads (Invitrogen) for 1 h at 4oC on rotation. Beads were washed sequentially with 300 µl of washing buffer I (20 mM of Tris/HCl, pH 8.0, containing 500 mM of NaCl, 0.1% of SDS and 2 mM of EDTA), 300 µl of washing buffer II (10 mM of Tris/HCl, pH 8.0, containing 250 mM of LiCl, 1 mM of EDTA, and 1% of dyoxyholate), and with 300 µl of Tris/EDTA buffer twice. Precipitated chromatin complexes were eluted with 100 µl of elution buffer (10 mM of DTT, 1% of SDS and 0.1 M of NaHCO3) for 15 min on shaker. Cross-linking was reversed by adding NaCl, to a final concentration of 200 mM and incubated at 65oC overnight. Remaining proteins were digested with the extraction buffer (50 mM of Tris/HCl, pH 6.8, containing 10 mM of EDTA, and 40 µg/ml of proteinase K) for 1 h at 45oC. DNA was recovered phenol / chloroform / isoamyl alcohol (25 / 24 / 1) extraction and ethanol precipitaded. Recovered DNA was blunt-ended with T4 DNA polymerase (Takara) before cloning into pBluescript SK vector. Sequencing was performed by Takara BIO INC.

Sequencing Platform

instrument_model
Illumina Genome Analyzer IIx

hg19

Number of total reads
22950398
Reads aligned (%)
77.0
Duplicates removed (%)
51.9
Number of peaks
2665 (qval < 1E-05)

hg38

Number of total reads
22950398
Reads aligned (%)
77.5
Duplicates removed (%)
51.2
Number of peaks
2750 (qval < 1E-05)

Base call quality data from DBCLS SRA