Antigen

Antigen Class

Input control

Antigen

Input control

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)

Sample

source_name

U2OS cells

tissue

U2OS cells

cell type

osteosarcoma

Sex

female

age

15 years

tissue/cells

bone

chip antibody

none

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 Genome Analyzer II

hg38

Number of total reads

35616776

Reads aligned (%)

97.2

Duplicates removed (%)

44.0

Number of peaks

1002 (qval < 1E-05)

hg19

Number of total reads

35616776

Reads aligned (%)

96.4

Duplicates removed (%)

46.7

Number of peaks

869 (qval < 1E-05)