RNAseq 0.5 hrs after VACV infection of HeLa Cells
. For the RNAseq experiment HeLa cells were infected with VACV and VACVE3L. The cells were lysed in RNAzol RT solution at 0.5, 1, and 2 hrs post infection. The RNA was extracted as mentioned earlier. RNA samples were qualified using the Agilent technologies 2100 Bioanalyzer (Total RNA Nano kit), all samples displayed RIN values >9, with the exception of VACdE3L-12-1 and VACdE3L-12-2. These samples displayed obvious 28S and 18S RNA peaks with some limited degradation that interfered with the RIN value calculation. RNA concentration was obtained from duplicate measurements using the QubitTM platform. Approximately 920 ng of RNA was used for library generation following the manufacturers protocol. After an initial poly-A selection step, the RNA was fragmented for 8 minutes, and converted to cDNA (1st and 2nd strand synthesis). 10nM stocks of each library were prepared using the average fragment length and concentration for each library. Pooled libraries (25 microlitres at ~2.6 nanograms/microlitre) were submitted for sequencing (2x125bp) on an Illumina HiSeq2500 at The Centre for Applied Genomics (TCAG), Toronto. The RNAseq data was analyzed using the Geneious for RNA seq' assembler in Geneious R9 version.