ShCtrl_pOHT
Homo sapiens
Protocols: U2OS DIvA cells were plated at 15cm and collected at 80% confluency   U2OS-DIvA (AID-AsiSI-ER-U2OS) cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), 100 unit/mL penicillin, 100 μg/mL streptomycin (Gibco), and 1 mM Sodium Pyruvate in the presence of 400 μg/mL G418.  To induce AsiSI-dependent double-strand breaks (DSBs), cells were treated with 300 nM (z)-4-Hydroxytamoxifen (4OHT) for 4h. Cells were fixed with 1% PFA for 10 minutes at room temperature, and cross-linking was stopped with 0.125 M Glycine for 5 min. After cell lysis, DNA was sheared to the size of 300–500 bp using a Vibra cell sonicator (15 sec ON, 30 sec OFF, 35% duty, 20 cycles). Five percent of each supernatant was used as input control and processed with the cross-linking reversal step. Chromatin was immunoprecipitated using 2ug of the indicated antibodies and Protein A/G magnetic beads. Following reverse cross-linking; the precipitated DNA was purified using the Macherey-Nagel Nucleospin kit NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, cat no. E7645), according to the manufacturers protocol, without fragmentation. ∼5 ng of purified DNA (average size 300-500 bp) was used for library preparation.
ENA first public
2025-04-17
INSDC center name
Department of Biology, Technion - Israel Institute of Technology
INSDC status
public
Submitter Id
E-MTAB-15055:ShCtrl_pOHT
broker name
ArrayExpress
cell line
U2OS
collection date
not collected
common name
human
developmental stage
juvenile stage
disease
osteosarcoma
genotype
AsiSI-ER expression
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
bone
sample name
E-MTAB-15055:ShCtrl_pOHT
scientific_name
Homo sapiens