title: ATACseqD_pOHT_24H_ATMi
organism: Homo sapiens
description: Protocols: ATAC-seq: Cells were washed twice with PBS and harvested with Trypsin and resuspended in 5ml of Dulbecco's modified Eagle's medium (DMEM). Cells were then counted and resuspended to have 5x105 cells/ml in 1X PBS. 1ml of suspension was then centrifuged at 600g for 5 minutes at room temperature and resuspended in 500ul ice cold PBS before being centrifuged again at 600g for 5 minutes at 4°C. 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. ATAC-seq: Nuclei were isolated by resuspending the cells in 500ul cold Lysis buffer (10mM Tris-HCl pH 7.5; 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween 20, 0.01% Digitonin) before being incubated on ice for 3 minutes. 10ml of cold lysis buffer without NP-40 & Digitonin was then added to stop the reaction. Nuclei were collected following centrifugation at 600g for 10 minutes at 4°C and resuspended in 165ul of ice cold PBS. 50000 cells (16.5ul) were then exposed to a transposition mix (25 µl 2X TD buffer (Illumina #15027866), 2.5 µl Transposase TDE1 (Illumina #15027865), 1720.1 µl Digitonin 5%, 0.5 µl Tween 20 10%, 5.4 µl H20) and incubated at 37°C for 30 minutes in a thermomixer at 700rpm. Transposed DNA was then purified using the Qiagen MinElute MinElute PCR Purification Kit (Cat. No. / ID: 28004) following manufacturers instructions. ATAC-seq: 20ul of transposed DNA was taken and 2.5 µL of 25 µM barcoded i7 and i5 primers were added together with 25 µL of NEBNext HiFi 2x PCR Master mix (New England Biolabs). Samples were subjected to PCR preamplification using the following conditions (72°C for 5 minutes, 98°C for 30 seconds then 5 cycles of 98°C for 30 s, 63°C for 10 s and 72°C for 1 minute). 4 µL of the preamplification reaction was taken and 0.5 µL of 25 µM barcoded i7 and i5 primers were added together with 6 µl Kapa HiFi Hot start realtime PCR mix (KM2702). To estimate the number of cycles required for PCR amplification, the reaction was subject to 20 cycles of real time PCR using the following cycling conditions (98°C for 30 seconds then 20 cycles of 98°C for 30 s, 63°C for 10 s, 72°C for 1 minute). The number of required cycles were determined by taking the cycle number needed for ¼ of the maximum fluorescence intensity. In this case, all samples were further subjected to PCR amplification using the following conditions (98°C for 30 seconds then 5 cycles of 98°C for 30 s, 63°C for 10 s, 72°C for 1 minute, followed by 72°C for 1 minute). Libraries were purified using Ampure XP as follows : large fragments were first removed with a 0.4X samples to beads ratio and the library was recovered with a 1.1X sample to beads ratio. Libraries were quantified with Quantus (Promega) and run on a High Sensitivity DNA chip on the Bioanalyzer 2100 instrument (Agilent Technologies).
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:ATACseqD_pOHT_24H_ATMi
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: ATACseq
attributes.tag: sample name
attributes.value: E-MTAB-15637:ATACseqD_pOHT_24H_ATMi
attributes.tag: scientific_name
attributes.value: Homo sapiens