Qiaseq stranded total rna lib kit

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.

Total RNA was extracted by using Direct-zol RNA MicroPrep (Zymo research, R2060). Library was prepared using QIAseq FastSelect RNA Removal Kit (Qiagen, 333180-24) and QIAseq Stranded Total RNA Lib Kit (Qiagen, 180743) following manufacturer’s instruction. In brief, 200–500 ng total RNA was used for each reaction. 1ul rRNA removal reagent was added into 28 μl of RNA sample, plus 5 μl 5 × RT Buffer, followed by incubation for 3 min at 95 °C, and then went through stepwise annealing using PCR programing. After fragmentation and rRNA removal, reverse transcription, second-strand synthesis, end-repair, A-addition, and strand-specific ligation with selected adapter (1:25 dilution) were performed, followed by CleanStart library amplification. Library DNA was then purified and size selected for around 500 bp fragments, and checked with Qubit and Agilent 4200 Tapestation.