ChIP-Atlas: SRX1810238

Antigen

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)

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: nuclei were extracted in swelling buffer, using Dounce homogenization, and diluted with sonication ChIP buffer to give a concentration of approximately 10 million cells per ChIP reaction in a volume of 1000 ul. Chromatin was sonicated to the average size of 200-400 bp. Reactions were performed in 1.5 ml sample tubes, using 40 ul of Protein G Dynabeads (Invitrogen) prebound with the appropriate antibody in each ChIP reaction. Washes were performed 3 times with Sonication buffer (50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1%SDS, 0.5 mM PMSF, Protease inhibitor cocktail (Roche)), 3 times with wash buffer A (Sonication buffer supplemented with 500mM NaCl), 3 times with wash buffer B (20 mM Tris, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF, Roche protease inhibitor cocktail] and TE buffer pH 8.0 (10 mM Tris-Cl pH 8.0, 1 mM EDTA pH 8.0 supplemented with 50 mM NaCl). 200 μl of elution buffer (50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS, 50mM NaHCO3) was added to the beads for 20 min at 65°C. Eluates were purified after reverse crosslink using QIAquick PCR Purification Kit (Qiagen). Libraries were prepared with the Illumina ChIP-seq Sample Prep Kit as per the manufacturer’s instructions, and sequenced on an Illumina HiSeq 2500 platform with 50bp single end reads.