Antigen

Antigen Class

No description

Antigen

NA

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

ENA first public

2015-07-16

ENA last update

2018-03-08

ENA-CHECKLIST

ERC000011

External Id

SAMEA2420691

INSDC center alias

Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom

INSDC center name

Centre for Gene Regulation and Expression, University of Dundee, Dundee DD1 5EH, United Kingdom

INSDC first public

2015-07-16T15:54:27Z

INSDC last update

2018-03-08T17:16:58Z

INSDC status

public

Submitter Id

E-MTAB-2368:growing_U2OS_cells_4

broker name

ArrayExpress

cell line

U2OS

cell type

human osteosarcoma cell

common name

human

individual

43_1

sample name

E-MTAB-2368:growing_U2OS_cells_4

sex

female

specimen with known storage state

fresh specimen

Sequenced DNA Library

library_name

heat_shock_input

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Human osteosarcoma (U2OS) cells were obtained from CRUK and grown at 37C according to standard cell culture protocols. U2OS cells were grown at 37C and either left untreated or incubated at 43C for 30 minutes to induce heat shock. Approximately 3.5-4.5 x 10 ^ 7 U2OS cells were used per treatment. Cells were chemically cross-linked by adding formaldehyde solution directly into cell culture medium to a final concentration of 1 %. Cells were fixed by incubation at room temperature for 10 min, followed by incubation with 0.125 M glycine for 5 min. Cells were washed twice and scraped into ice-cold PBS. Cell pellets were flash frozen in liquid nitrogen and stored at -80C. Frozen cell pellets were lysed in 2.1 ml lysis buffer containing 1% SDS, 10 mM EDTA, 50 mM Tris (pH 8.1), and protease inhibitors. To shear chromatin to fragments of about 200-500 bp size, samples were sonicated in 300 _l volumes for 14 cycles (7.5 min total sonication time) at high setting using a Bioruptor (Diagenode). Sonicated lysates were then cleared by centrifugation for 10 min at high speed, diluted 1/10 with dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris at pH 8.1) and incubated with SUMO-2 antibody (Life Technologies, 51-9100) and 0.1% Brij-35 overnight at 4C. In detail, to capture protein-DNA complexes, a fraction corresponding to 90% of the lysate volume was incubated with 21 ug SUMO-2 antibody and subsequently with protein G sepharose 4B beads (Sigma) for 1 h using a final bead bed volume of 150 ul. To reduce non-specific binding, beads had been pre-incubated with 0.5% (w/v) BSA in PBS overnight. Libraries were constructed using approximately 10 ng purified DNA. Single-end libraries were generated using either the Illumina ChIP-seq DNA sample Preparation Kit (IP-102-1001) or the TruSeq DNA sample preparation kit (PE-940-2002). In brief, ChIP or input DNA was end repaired and A-tailed before adaptors were ligated on the fragments. The ligated samples were then size selected on a 2 % low melting agarose gel to approximately 250-300 bp. An 18 cycle PCR was performed and libraries were quantified using a Nanodrop Spectrophotometer. Library quality was validated by High Sensitivity Bioanalyzer ChIP (Agilent).

Sequencing Platform

instrument_model

Illumina Genome Analyzer IIx

hg38

Number of total reads

31532398

Reads aligned (%)

97.3

Duplicates removed (%)

27.3

Number of peaks

915 (qval < 1E-05)

hg19

Number of total reads

31532398

Reads aligned (%)

96.5

Duplicates removed (%)

28.7

Number of peaks

991 (qval < 1E-05)