characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2016-01-01T17:01:19Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:30:45Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2497031
characteristics.characteristic: External Id
characteristics.text: Division of Cancer Biology, Institute of Cancer Research
characteristics.characteristic: INSDC center name
characteristics.text: 2016-01-01T17:01:19Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:30:45Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS449959
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2550:EXOSC8i_replicate_2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: MDA-MB-231
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001209
characteristics.characteristic: cell line
characteristics.text: breast adenocarcinoma
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000304
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: cells were grown in normal DMEM with 10% FBS in standard tissue culture conditions. Cells were transfected with 10nM final concentration of non-targetting vs EXOSC5, EXOSC6, EXOSC8, or EXOSC9 siGENOME smartpool siRNAs, using Lipofectamine RNAiMax according to manufacturer's instructions. 72 hr later, cells were washed with PBS and then lysed in RLT buffer from Qiagen's RNeasy Mini Kit. RNeasy Mini Kit (Qiagen) was used for RNA purification. total RNA quality was assessed using the 2100 Bioanalyzer (Agilent). In case of DNA contamination, DNA levels were first estimated by RT-PCR. If DNA contamination was above 2%, the sample was cleaned up using RNeasy Plus Micro Kit (Qiagen). Poly-A mRNA (1-5% of total RNA) was then isolated from the total RNA sample using the Dynabeads mRNA Purification Kit (Life Technologies). Purified mRNAs were fragmented using the mRNA Fragmentation Reagents (Life Technologies) and ethanol precipitated. The size range of generated fragments was kept at 60 to 1000 nucleotides, with a peak at around 150 nucleotides. To make cDNA from the fragmented RNA samples, NEBNext mRNA Library Prep Reagent Set for Illumina was used (NEB), with the synthesis being performed Strand-wise (first-strand synthesis then second-strand synthesis) followed by purification using QIAquick Columns (Qiagen). End Repair, dA-tailing, and Adapter ligation were all performed based on NEBNext mRNA Library Prep Reagent Set for Illumina instructions. Agarose gel separation was used to select for the appropriate library size. To get a library size of ?400bp (insert size ?330bp) a 350-500bp slice was taken from each of the samples. Gel extraction was then performed using Qiagen MinElute gel extraction kit (Qiagen). To enrich for the correctly adapter-ligated and size-selected cDNA, a PCR was performed using the Herculase II polymerase enzyme (Agilent), also adding the specific indexes for multiplexing. The PCR was then followed by an AMPure bead purification to exclude primer-dimers and other smaller fragments from the final library. The quantity and quality of the prepared library was evaluated on Bioanalyzer and the samples were quantified using qPCR. Subsequently, libraries were combined for multiplexed sequencing runs.
characteristics.characteristic: description
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: EXOSC8i_replicate_2
characteristics.characteristic: title