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
ERS27132928
SRA accession
E-MTAB-12712_2:HiC_siCTRL_mOHT
Submitter Id
ArrayExpress
broker name
U2OS
cell line
not collected
collection date
human
common name
Protocols: For Hi-C: 10x106 cells were harvested by trypsin and resuspend in PBS. Formaldehyde was added at a final concentration of 2% and crosslinking was allowed to proceed for 10 min at room temperature. Reaction was stopped by adding Stop solution from Arima Hi-C kit (Arima Genomics) and incubation for 5 minutes at room temperature then incubate for 15 min on ice. Cells were resuspended in PBS and 1x106 cells per aliquot were freeze in liquid nitrogen.  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)  Hi-C experiments were performed in DIvA cells using the Arima Hi-C kit (Arima Genomics) according to the manufacturer’s instructions. 1 million cells were used by condition. Briefly, cells were cross-linked with 2% formaldehyde for 10 min at RT, lysed and chromatin was digested with two different restriction enzymes included in the kit. Ends were filled-in in the presence of biotinylated nucleotides, followed by subsequent ligation. Ligated DNA was sonicated using the Covaris S220 to an average fragment size of 350 bp with the following parameters (Peak incident power: 140; Duty factor: 10%; Cycles per burst: 200; Treatment time: 70s). DNA was then subjected to a double-size selection to retain DNA fragments between 200 and 600 bp using Ampure XP beads (Beckman Coulter). Biotin-ligated DNA was precipitated with streptavidin-coupled magnetic beads (included in the kit). Hi-C library was prepared on beads using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina and NEBNext® Multiplex Oligos for Illumina (New England Biolabs) following instructions from the Arima Hi-C kit.
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
HiC_siCTRL_mOHT
title