ferret_485_Spleen
Following the manufactures protocols, 800 ng of total RNA was used as input material in to the QIAseq FastSelect rRNA (HMR) (Qiagen) protocol to remove cytoplasmic and mitochondrial rRNA with a fragmentation time of 7, 13 or 15 minutes. Subsequently the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to generate the RNA libraries. Following 8 cycles of amplification the libraries were purified using Ampure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Fragment analyser. Final libraries were pooled in equimolar amounts using the Qubit and Bioanalyzer data. The quantity and quality of each pool was assessed by Bioanalyzer and subsequently by qPCR using the Illumina Library Quantification Kit from Kapa (KK4854) on a Roche Light Cycler LC480II according to manufacturer's instructions. Briefly, a 10 l PCR reaction (performed in triplicate for each pooled library) was prepared on ice with 6 l SYBR Green I Master Mix and 2 l diluted pooled DNA (1:1000 to 1:100,000 depending on the initial concentration determined by the Qubit dsDNA HS Assay Kit). PCR thermal cycling conditions consisted of initial denaturation at 95C for 5 minutes, 35 cycles of 95C for 30 seconds (denaturation) and 60C for 45 seconds (annealing and extension), melt curve analysis to 95C (continuous) and cooling at 37C (LightCycler LC48011, Roche Diagnostics Ltd, Burgess Hill, UK). Following calculation of the molarity using qPCR data, template DNA was diluted to 300pM and denatured for 8 minutes at room temperature using freshly diluted 0.2 N sodium hydroxide (NaOH) and the reaction was subsequently terminated by the addition of 400mM TrisCl ph=8. To improve sequencing quality control 1% PhiX was spiked-in.