title: RNA-SEQ_siSETX_mOHT
organism: Homo sapiens
description: Protocols: 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. For AsiSI-dependent DSB induction, cells were treated with 300 nM OHT (Sigma; H7904) for 4h or 24h. For ATM inhibition, cells were pretreated for 1 h with 20μM KU-55933 (Sigma, SML1109) and during subsequent 4OHT treatment. For DNA-PK inhibition, cells were pretreated for 1 h with 2μM NU-7441 (Selleckchem, S2638) and during subsequent 4OHT. 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 sequences: CTRL: CAUGUCAUGUGUCACAUCU; or SCC1: GGUGAAAAUGGCAUUACGG: or SMC1 (siSMC1): UAGGCUUCCUGGAGGUCACAUUUAA; or 53BP1 (si53BP1): GAACGAGGAGACGGUAAUA; or SUN2 (siSUN2): CGAGCCTATTCAGACGTTTCA; or ARP2 (siARP2): GGCACCGGGUUUGUGAAGU or SETX (siSETX): GAGAGAAUUAUUGCGUACU. For cell synchronization, cells were incubated for 18 h with 2 mM thymidine (Sigma, T1895), then released during 11 h, followed by a second thymidine treatment for 18 hr. S, G2 and G1 cells were then respectively treated with OHT at, 0, 6 or 11 h following thymidine release and harvested 4 h later. RNA-seq library preparation were performed at EMBL Genomics core facilities (Heidelberg, Germany) using TruSeq Stranded Total RNA (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-25
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-25
attributes.tag: External Id
attributes.value: SAMEA112245945
attributes.tag: INSDC center alias
attributes.value: MCD, UMR5077, CBI
attributes.tag: INSDC center name
attributes.value: MCD, UMR5077, CBI
attributes.tag: INSDC first public
attributes.value: 2022-12-25T00:22:01Z
attributes.tag: INSDC last update
attributes.value: 2022-12-25T00:22:01Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10865_2:RNA-SEQ_siSETX_mOHT
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: U2OS
attributes.tag: cell_type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: juvenile stage
attributes.tag: disease
attributes.value: osteosarcoma
attributes.tag: genotype
attributes.value: AsiSI-ER
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bone
attributes.tag: protocol
attributes.value: RNA
attributes.tag: sample name
attributes.value: E-MTAB-10865_2:RNA-SEQ_siSETX_mOHT