Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
RELA

Cell type

Cell type Class
Bone
Cell type
U2OS
Tissue
bone
Lineage
mesoderm
Description
osteosarcoma from the tibia of a 15 year old, J. Ponten and E. Saksela derived this line (originally 2T) in 1964 from a moderately differentiated sarcoma, viruses were not detected during co-cultivation with WI-38 cells or in CF tests against SV40, RSV or adenoviruses, mycoplasma contamination was detected and eliminated in 1972, (PMID: 6081590)

Attributes by original data submitter

Sample

source_name
U2OS cells
tissue
U2OS cells
cell type
osteosarcoma
Sex
female
age
15 years
tissue/cells
bone
chip antibody
NFKB p65 polyclonal Ab (Diagenode, C15310256)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
2x10^6 cells were inoculated on 10 cm2 dish 2 days before experiments. At the appointed experimental points a two-step cross-linking procedure was applied for an efficient fixation of NF-κB dimers with DNA [27]. Cells were washed twice with PBS, a solution of DSG (disuccinimidyl glutarate) in PBS was added to the final concentration of 2 mM, and then cells were incubated for 45 minutes at RT with rotation. Next, cells were washed twice with PBS and the second fixation was performed using 1% formaldehyde for 10 min at RT. Formaldehyde fixation was quenched by glycine (125 mM final concentration) and then nuclei were isolated using buffers and protocol from iDeal ChIP-seq Kit for Transcription Factors (Diagenode). Chromatin of nuclei re-suspended in 200 µl was sheared using Bioruptor® PLUS combined with the Bioruptor® Water cooler & Single Cycle Valve (at HIGH power setting) with 15 cycles of 30 sec shearing followed by 30 sec of standby; chromatin fragments with approximate length 100-600 bp were obtained. Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode) with 3µl/sample of anti-RelA(p65) polyclonal antibody (C15310256, Diagenode) or without antibody (mock-IP), according to the manufacturer protocol. Immunoprecipitated DNA fragments and input DNA were sequenced using the HiSeq 1500 system with TruSeq workflow (Illumina)

Sequencing Platform

instrument_model
Illumina HiSeq 1500

hg19

Number of total reads
54916386
Reads aligned (%)
98.0
Duplicates removed (%)
7.5
Number of peaks
3041 (qval < 1E-05)

hg38

Number of total reads
54916386
Reads aligned (%)
98.9
Duplicates removed (%)
5.9
Number of peaks
3355 (qval < 1E-05)

Base call quality data from DBCLS SRA