characteristics.text: 2014-05-29T17:02:19Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:33:08Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2146551
characteristics.characteristic: External Id
characteristics.text: University College London
characteristics.characteristic: INSDC center name
characteristics.text: 2014-05-29T17:02:19Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:33:08Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS317034
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1717:KC2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: HEKn
characteristics.characteristic: cell line
characteristics.text: keratinocyte
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000312
characteristics.characteristic: cell type
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Neonatal primary human epidermal keratinocytes (HEKn, Life technologies, Paisley, UK) were cultured on mouse collagen IV (0.67ug/cm2 BD Biosciences, Oxford, UK) coated surfaces in keratinocyte defined media containing epithelial growth factor (KDM and EpiLife, Life technologies). Differentiation was induced by shifting the cells to a high calcium media containing [1.2mM] Calcium Chloride. Viral infections for transcriptome experiments were carried out using VZV pOka cultured and used at less than 20 passages. Infected cell preparations were first treated with Mitomycin C (0.05mg/ml for 3hrs) prior to freezing and subsequently titered on Mewo cells. Cell-free VZV was generated by a rapid freeze (liquid N2) and thaw (37C), followed by removal of cellular debris by low speed centrifugation. For UV inactivated VZV, cell-free VZV supernatants were treated for 20 min at 150,000J/Cm2 using a Stratalinker UV Crosslinker (Stratagene). 1x105 HEKn cells per well of a 6 well dish were inoculated with 3x104 infectious units of virus. All VZV cultures were carried out at 34C, 5% CO2. This method involves several steps that are designed to convert total RNA into a library of template molecules suitable for high throughput DNA sequencing. The first step involves purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved mRNA fragments are copied into first strand cDNA using reverse transcriptase and a high concentration of random hexamer primers. Unique adaptors were ligated to the cDNA and 200bp fragments were size selected by agarose gel purification. Libraries were validated by using a DNA high sensitivity chip (Agilent, Cheshire, UK) and quantified by Qubit analysis using the Quant-iT dsDNA HS Assay (Life technologies).
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: KC2
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0002068
characteristics.characteristic: title