2025-11-13
ENA first public
ERC000011
ENA-CHECKLIST
MCD, Centre de Biologie Intégrative (CBI), CNRS, Université de Toulouse, UT3
INSDC center name
public
INSDC status
ERS27132924
SRA accession
E-MTAB-12712_2:ChIP-seq_SUN1_pOHT_siCTRL
Submitter Id
ArrayExpress
broker name
U2OS
cell line
not collected
collection date
human
common name
Protocols:  For ChIP: Formaldehyde was added to the culture medium at a final concentration of 1% and crosslinking was allowed to proceed for 15 min at room temperature. Reaction was stopped by adding glycine (0.125M final concentration) and incubation for 5 minutes at room temperature. Cells were washed twice with PBS and harvested by scraping.  DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (Invitrogen) with 1 µg/mL puromycin (DIvA cells) at 37°C under a humidified atmosphere with 5% CO2. For AsiSI-dependent DSB induction, cells were treated with 300 nM OHT (Sigma; H7904) for 4h. siRNA transfection were performed using the 4D Nucleofector X (kit SE cell line) according to the manufacturer's instructions and subsequent treatment(s) were performed 48 h later. siRNA: CTRL: siGENOME Non-targeting Control #2 Dharmacon (D-001206-13), siPER2 Dharmacon (M-012977-01) For ChIP: 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 (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). Antibodies: PER2 Proteintech 20359-1-AP (5µg), BMAL1 Abcam ab3350 (2µg), SUN1 Sigma HPA008346 (5µg), SUN2 Abcam ab124916 (5µg). For ChIP: Sequencing libraries were prepared from IP of PER2, BMAL1, SUN1 and SUN2 samples using the NEBNext® Ultra™ II Library Prep Kit (NEB)for Illumina.
description
osteosarcoma
disease
AsiSI-ER
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
bone
organism part
Homo sapiens
scientific_name
ChIP-seq_SUN1_pOHT_siCTRL
title