ATAC_3
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-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. Cell were not treated  Cell lysis was performed by incubation in cell lysis buffer (5 mM Pipes pH 8, 85 mM KCl, 0.5% IGEPAL® CA-630) followed by Dounce homogenization. Nuclei were harvested by centrifugation and incubated in nuclear lysis buffer: (50 mM Tris pH 8.1, 10 mM EDTA, 1% SDS) and sonicated 10 times for 10 s at a power setting of 5 and 50% duty cycle (Branson Sonifier 250). Samples were diluted 10 times in ChIP dilution buffer (16.7 mM Tris pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X‐100, 0.01% SDS) and precleared using a mixture of protein A-agarose (Pierce) and protein G-sepharose (Sigma) previously blocked with BSA (Sigma). Precleared samples (10 to 200 μg) were incubated overnight at 4°C with indicated antibodies. Immune complexes were recovered by incubation with blocked protein A/protein G beads for 2 h at 4°C on a rotating wheel. Beads were washed once in dialysis buffer (50 mM Tris pH 8, 2 mM EDTA, 0.2% Sarkosyl), five times in wash buffer (100 mM Tris pH 8.8, 500 mM LiCl, 1% IGEPAL® CA-630, 1% NaDoc) and twice with TE (10mM Tris-HCl, pH 8, 1mM EDTA). Beads were resuspended in TE supplemented with 50µg/ml DNase-free RNase A (Abcam) and incubated for 30 minutes at 37°C. SDS (final concentration 0.5%) was added and crosslink was reversed by overnight incubation at 70°C. Following a 2 hours treatment with 200 µg/ml proteinase K (Roche) at 45°C, DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation in presence of 5 µg glycogen (Invitrogen). DNA pellet was resuspended in water. Prior to next-generation sequencing library preparation, samples from multiples ChIP experiments were pooled and sonicated for 5 cycles (30 seconds on, 30 seconds off, high setting) on a Bioruptor (Diagenode) then concentrated with a vacuum concentrator (Eppendorf). 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).
ENA first public
2026-07-31
INSDC center name
CBI, MCD, University of Toulouse, FRANCE
INSDC status
public
Submitter Id
E-MTAB-15750:ATAC_3
broker name
ArrayExpress
cell line
U2OS
cell type
epithelial cell
collection date
not collected
common name
human
disease
osteosarcoma
genotype
AsIS1-ER
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
bone
sample name
E-MTAB-15750:ATAC_3
scientific_name
Homo sapiens