VZV infection of primary keratinocytes
VZV infection of primary keratinocytes
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).
University College London
E-MTAB-1717:KC4
Experimental Factor: developmental stage
Differentiated
Experimental Factor: disease
normal
Experimental Factor: individual
Run2_Lane3