characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-04-20T00:13:57Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-04-20T00:13:57Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110585833
characteristics.characteristic: External Id
characteristics.text: NTU
characteristics.characteristic: INSDC center name
characteristics.text: 2023-04-20T00:13:57Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-04-20T00:13:57Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12683618
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12059:siJMYC
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: U2OS
characteristics.characteristic: cell_line
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Cells were treated with Non-targeting control or JMY siRNA for 72h, the last 6h cells were treated with 50uM etoposide before harvesting Cells were routinely grown in DMEM (high glucose, supplemented with glutamax) with 5% foetal bovine serum and no antibiotics. 72h siRNA treatment with last 6h 50uM etoposide treatment Sequencing libraries were generated using NEBNext® Ultra™ RNA LibraryPrep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand SynthesisReaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected,adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. ThenPCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
characteristics.characteristic: description
characteristics.text: osteosarcoma
characteristics.characteristic: disease
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: bone
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: etoposite; 50 micromolar
characteristics.characteristic: stimulus
characteristics.text: siJMYC
characteristics.characteristic: title