*2.9. 3mRNA Sequencing*

A549 wt, RIG-I KO (clone B05) and MDA5 KO (clone c27) cells were lysed at 24 h post infection with ZIKV (multiplicity of infection (MOI): 5), and total RNA was extracted using the QIAshredder (Qiagen) and RNeasy Mini Kit (Qiagen) according to manufacturer's instructions. RNA concentration was measured by the Qubit RNA HS (high sensitivity) Assay Kit (Life Technologies) according to the manufacturer's instructions. Quality of the extracted RNA was controlled by the Agilent 2200 TapeStation System (Agilent Technologies).

cDNA was generated from total RNA using first oligo-dT and subsequently random priming. The prepared libraries were QC'ed and multiplexed before sequencing over one lane of the NextSeq flow cell (high output, 75 bp single reads).

Following QC analysis with the fastQC package, reads were aligned using STAR against the human genome assembly (GRCh38 (hg38) UCSC transcripts) [27]. Read counts were visualized using UCSC genome browser [28]. Gene expression levels were quantified as read counts using the featureCounts function from the Subread package with default parameters [29]. The read counts were used for the identification of global differential gene expression between specified populations using the edgeR package [30]. RPKM values were also generated using the edgeR package [30]. Genes

were considered differentially expressed between populations if they had an adjusted *p*-value (false discovery rate, FDR) of less than 0.05. The edgeR package was also used to generate heatmaps and plots [30]. The Venn diagram was created using [31].
