Illumina-based RNA-seq workflow

RNA was extracted from dry cell pellet (~1.5E6 cells) according to the High Pure RNA Isolation Kit (Roche, Penzberg, Germany) manual. For Illumina-based sequencing, samples were prepped using TruSeq Stranded mRNA Library Preparation Kit according to TruSeq Stranded mRNA Sample Preparation Guide. Sequencing was performed on an Illumina HiSeq 4000 (Illumina, San Diego, California, USA) using run mode SR50. Reads were trimmed using sickle-1.33 (Joshi and Fass, 2011 ) and aligned to hg19 using hisat2-2.0.4 (Kim et al., 2015 (link)). The alignments were assigned to genes and exons using featurecount-1.5.0-p3 (Liao et al., 2014 (link)) using the gene annotation provided by the iGenomes resource (Illumina, 2020 ). For quantitative RT-PCR we used Biorad iScript cDNA Synthesis Kit and LightCycler 480 FastStart DNA Master SYBR Green I (Roche). Measurements were performed with LightCycler 480 System and corresponding software (Roche). To determine the quantitative gene expression data levels were normalized to the geometric mean of two housekeeping genes. All experiments were at least performed in duplicate with three technical replicates per experiment. Primer sequences used in this study are:
Both ISG15 and IFIT1 primers sequences were derived from previous studies (Bektas et al., 2008 (link); Labbé et al., 2012 (link)).