RNA-seq Analysis of Differentially Expressed Genes

RNA sequencing and analysis of the data for obtaining differentially expressed genes were described in a separate work [35 ]. Briefly, RNA was extracted with NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany) as described previously [13 ]. RNA-seq libraries were prepared using QIAseq stranded Total RNA Lib kit (Qiagen) and were sequenced using NextSeq 500 (Illumina). It ended up 76 base pair (bp) single-end reads. Quality and rRNA filtering was performed using Trimmomatic v0.36 [60 (link)] and SortmeRNA v2.1b [61 (link)]. The reads were mapped to ScottA genome (GenBank: CM001159.1) using Bowtie2 [62 (link)]. HTseq v2.3.4.3 [63 (link)] was used to obtain raw gene counts. Raw counts were normalized and pairwise differential expression analysis between control and treated samples was performed using DESeq2 [64 ]. The threshold for differentially expressed genes was set adjusted, p-value ≤0.05 and log2 fold change (log2 FC) ≥0.6. Normalized read counts and log2 FC data were used for analysis. RNA-seq data is available in the European Nucleotide Archive (ENA) under accession code PRJEB34771.