title: CTRLi_replicate_2
organism: Homo sapiens
description: 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 EXOSC8 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2016-01-01T17:01:19Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T17:30:56Z
attributes.tag: External Id
attributes.value: SAMEA2497033
attributes.tag: INSDC center name
attributes.value: Division of Cancer Biology, Institute of Cancer Research
attributes.tag: INSDC first public
attributes.value: 2016-01-01T17:01:19Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T17:30:56Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2550:CTRLi_replicate_2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: MDA-MB-231
attributes.tag: cell type
attributes.value: breast adenocarcinoma
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-2550:CTRLi_replicate_2
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: specimen with known storage state
attributes.value: fresh specimen