Photoscript 2 first strand cdna synthesis kit

The transcriptome data was validated by selection of at least 8 DEGs of top up-regulated and down-regulated from each comparison in the nymph, unfed and partly-fed adult tick transcriptome libraries by RT-qPCR. The cDNA was synthesized from aliquots of the RNA samples that were previously shipped for RNA-seq along with their corresponding biological duplicates using PhotoScript II First Strand cDNA Synthesis Kit (New England BioLabs Inc, Ipswich, US) according to manufacturer´s instruction. The qPCR reactions were performed using Luna Universal qPCR Master Mix (New England BioLabs Inc) according to manufacturer’s protocol. Primers for qPCR assay were designed by first locating the open-reading frame (ORF) of the DEG using the NCBI open reading frame finder followed by Primer-BLAST tool of NCBI [50 (link)]. The list of primers generated for the validation of RNA-seq data is presented in S1 Tables. E6rcfvTGB/Hnjach reaction was performed in three technical replicates and the A. hebraeum translation elongation factor 1 alfa (GenBank accession number AF240836) was used as a reference gene for normalization of the qPCR assays. The expression levels of the selected DEGs were calculated using the 2^–∆∆Ct method, followed by the comparison of Log₂fold change between RNA-seq and qPCR [51 (link)].

RNA was extracted from the challenged PL at the moribund stage using QIAgen’s QIAamp Viral RNA mini kit according to the protocol of manufacturer. The purity and concentration of the extracted RNA were assessed by Nanodrop-2000 spectrophotometer. The extracted RNA was reverse-transcribed to cDNA using New England Biolab’s cDNA kit (PhotoScript II First Strand cDNA Synthesis Kit, New England Biolabs, Ipswich, MA, USA).