Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K9me3

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
cell
U2Os cells
treatment
Control
antibody
anti H3K9me3 abcam ab8898

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 1% formaldehyde for 8 min, quenched with 1.25 M glycine, washed in PBS. Cells were resuspended in cells lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, 1x Roche Complete Protease inhibitor), kept in ice for 10 min, and sonicated using Bioruptor. Following sonication insoluble material was pelleted by centrifugation at 16000g for 10 minutes at 4°C. A small sample of soluble crosslinked chromatin was purified, checked for correct sonication size range. 25 ug of chromatin was diluted to 350ul with RIPA buffer (10 mM Tris pH 7.5, 140 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate, 1x Roche Complete Protease inhibitor). 10 μl of Protein A Dynabeads (Invitrogen) prebound with 2 μg of histone marks antibodies were added. Samples were incubated overnight at 4°C with rotation. Beads were precipitated on magnet stand and washed 3 times with 1 mL of RIPA buffer and once with TE. Crosslink was reversed with 250 μl of elution buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 5 μg/mL proteinase K) at 65°C for 2 hours. DNA was purified with Qiagen PCR cleanup kit. Concentration of eluted chromatin was measured with Qubit dsDNA HS Assay Kit (Life Technology). Sequencing libraries were prepared using the Illumina TruSeq DNA Sample Preparation Kit according to the manufacturer's protocol. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250-450 bp (insert plus adaptor) were band isolated from an agarose gel. DNA fragments were sequenced using paired-end sequencing technology on Illumina HiSeq 2000 platform.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
68026832
Reads aligned (%)
96.3
Duplicates removed (%)
42.8
Number of peaks
15582 (qval < 1E-05)

hg19

Number of total reads
68026832
Reads aligned (%)
93.2
Duplicates removed (%)
45.3
Number of peaks
12222 (qval < 1E-05)

Base call quality data from DBCLS SRA