ChIP-Atlas: SRX3586582

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: U2OS-GRα cells grown on 15-cm dishes were transfected with the appropriate siRNAs. After 48 h, the cells were treated with 100 nM dex for 1 h before crosslinking with 1% (v/v) formaldehyde for 10 mins at room temperature and extracting chromatin from the harvested cells. Chromatin was sonicated 20-30 min (30s on/off cycles) with the Biorupter (Diagenode) at 4ºC to produce DNA fragment size of 400-600 bp. Immunoprecipitation of the sonicated chromatin samples was conducted with a cocktail of GR antibodies: H300 (6 µg, Santa Cruz Biotechnology), PA1-511A (2 µg, Thermo Scientific), D6H2L (2 µg, Cell Signaling Technology) in a volume of 1 ml containing chromatin from 20 x 106 cells. Protein G Sepharose magnetic beads (GE Healthcare) were used to isolate the immune complexes with the crosslinked DNA. Quality of the DNA was assessed using Agilent Technologies 2100 Bioanalyzer and RT-qPCR analysis was performed for selected GR binding sites as quality control. Standard illumina protocols were used to construct the library for sequencing.