title: ChIPseq_IgG_pOHT_4H
organism: Homo sapiens
description: Protocols: ChIP: cells were crosslinked with 1% formaldehyde for 15 min at room temperature and quenched with 0,125 M glycine for 5 min. Cells were washed with 1X PBS and harvested by scrapping. DIvA (AsiSI-ER-U2OS) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) and 1µg/mL puromycin at 37°C under a humidified atmosphere with 5% CO2. AsiSI-dependent DSBs were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h. Inhibitors used were added to the medium 45 minutes prior to 4-OHT treatment. siRNA transfections were performed using 4D-Nucleofector from Lonza either with the SE-small kit for 1 million cells or SE-large kit for 5-20 million of cells, according to the manufacturer's instruction. Cells were collected 48 h after siRNA transfection. ChIP: Cell pellets were resuspended in cellular lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% NP-40)and homogenized with a Dounce homogenizer. Nuclei were harvested by centrifugation and resuspended in nuclear lysis buffer (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated using a Branson Sonifier 250 (at a power setting of 5 and 50% duty cycle) to obtain fragments of about 500 bp. Samples were diluted 10 times in dilution buffer (16.7 mM Tris pH 8, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) and each aliquot of 200 µg of chromatin was precleared with 100 µL of 50% protein-A (Pierce) and protein-G (Sigma) beads, (previously blocked with 500 µg of BSA) during 2 hours at 4°C on a rotating wheel. 100 µL of pre-cleared chromatin was kept at -20°C as input sample and 10-200 µg was incubated with specific antibodies at 4°C on a rotating wheel overnight. The next day, 100 µL of pre-blocked protein A/G beads were added to each sample for 2 hours at 4°C on a rotating wheel. Beads were washed once with dialysis buffer (50 mM Tris pH 8.1, 2 mM EDTA, 0.2% Sarkosyl), five times with wash buffer (100 mM Tris pH 8.8, 500mM LiCl, 1% NP-40, 1% NaDoc) and twice in TE buffer (Tris 10 mM pH8, 1 mM EDTA). Immunoprecipitated complexes were resuspended in 200 µL of TE and input samples were adjusted to 200 µL. Next, 0.5 µL of RNase A (30mg/mL) was added during 30 min at 37°C. Following addition of 0.5% SDS, samples were incubated overnight with shaking at 70°C to reverse the crosslink. Next, proteinase K was added (200 µg/mL final) and samples were incubated for 1h30 at 45°C. DNA was purified by phenol:chlorophorm extraction followed by ethanol precipitation. Samples were resuspended in 100 µl water. ChIP: multiple ChIP experiments were pooled, sonicated for 7 cycles (30 sec on, 30 sec off, high setting) with a Bioruptor (Diagenode), then concentrated with a vacuum concentrator (Eppendorf). About 10 ng of purified DNA was subjected to library preparation (NEBNext Ultra II DNA Library kit, New England Biolabs)
attributes.tag: ENA first public
attributes.value: 2026-04-18
attributes.tag: INSDC center name
attributes.value: MCD-CBI, University of Toulouse, Toulouse, France
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15637:ChIPseq_IgG_pOHT_4H
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: U2OS
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: osteosarcoma
attributes.tag: genotype
attributes.value: AsiS1-ER
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bone
attributes.tag: protocol
attributes.value: ChIPseq
attributes.tag: sample name
attributes.value: E-MTAB-15637:ChIPseq_IgG_pOHT_4H
attributes.tag: scientific_name
attributes.value: Homo sapiens