After extraction by Trizol reagent (Thermo Fisher Scientific) RNA was purified by the Direct-zol RNA kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s instructions. Quality analysis of RNA samples was performed on a fragment analyzer (Advanced Analytical Technologies (Agilent), Ankeny, IA, USA). RNA of samples with a RIN (RNA integrity number as a means of quality assessment) equal to 7 or greater was further processed and hybridized to Illumina HT-12 v4 Expression BeadChips (Illumina, San Diego, CA, USA) and measured on the Illumina HiScan. Expression data was processed through SOM machine learning as described in our previous publication on rubella virus-infected iPSC lines [24 (link)]. Briefly, we applied the R-program “oposSOM” [64 (link)] which generates images of the transcriptomic landscape of each sample revealing clusters of over- and under-expressed genes as red and blue spot-like areas. Their functional context was evaluated by means of gene set analysis as implemented in [65 (link)].
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