2025-06-12
ENA first public
ERC000011
ENA-CHECKLIST
MCD, Centre de Biologie Intégrative (CBI), CNRS, Université de Toulouse, UT3
INSDC center name
public
INSDC status
ERS22948348
SRA accession
E-MTAB-13197_2:qDRIP_mOHT
Submitter Id
ArrayExpress
broker name
U2OS
cell_line
epithelial cell
cell_type
not collected
collection date
human
common name
Protocols: qDRIP-seq was adapted from Crossley et al, NAR, 2020. In brief, 2.5 × 10 6 of trypsinized DIvA cells were mixed to 1.67 × 10 6 Drosophila S2 cells DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogenInvitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. Total RNA spike-ins were prepared from mid-log phase S. cerevisiae (BY4741 strain) cells grown in YPD + 2% glucose and labeled with 5 mM 4-thiouracil (4TU, Sigma-Aldrich, 440736) for 5 min. AsiSI dependent-DSB were induced by the addition of 300 nM 4-OHT (Sigma, H7904) into the medium for 4 h or 24 h. Etoposide (Sigma, E1383) was used at 50 µM for 30 min for END-seq, 50 µM for 1 h for ChIP. 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 qDRIP-seq was adapted from Crossley et al, NAR, 2020. In brief, 2.5 × 10 6 of trypsinized DIvA cells were mixed to 1.67 × 10 6 Drosophila S2 cells and lysed overnight at 37°C in TE buffer containing 0.5% SDS and 800 μg proteinase K (Roche, 03115828001). DNA was extracted by phenol:chloroform extraction using phase lock tubes (Qiagen, MaxTract, 129065) followed by ethanol precipitation. DNA was resuspended on ice in 130 μl TE buffer before sonication in Covaris S220 (microtubes PN520045) to obtain ~300 bp DNA fragments (Covaris S220, 140W peak incident power, 10% Duty factor, 200 cycles per burst for 80 sec). Immunoprecipitation was performed in triplicate by incubating 4 μg of sonicated DNA with 10 μg of S9.6 antibody (Antibodies Incorporation) in 1x binding buffer (10 mM NaPO 4 , 140 mM NaCl, 0.05 % Triton X100) overnight and at 4°C. Agarose A/G beads (Thermo Scientific, 20421) we added to samples for 2 h at 4°C. Beads were washed 4 times in binding buffer followed by incubation with elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, 0.3 μg/μL proteinase K) for 45 min at 55°C. Samples were subjected to phenol:chloroform extraction and ethanol precipitation, and were resuspended in low EDTA TE.  Sequencing libraries were prepared using the Swift ACELL-NGS 1S Plus kit according to the manufacturer’s instructions using 12 PCR cycles. Libraries were pooled at equimolar concentrations.
description
osteosarcoma
disease
AsiS1-ER
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
bone
organism part
qDRIP-seq
protocol
Homo sapiens
scientific_name
qDRIP_mOHT
title