title: RNA-Seq of PKD infected Salmo trutta (juvenile) left pelvic fin pool (pooling of RNA extracts)
design: RNA, extracted from the left pelvic fins of seven brown trout samples (July fin pool; sample IDs: J60, J73, J87, J89, J115, J137, J154), was pooled before the library preparation. Library preparation was done according to Illumina TruSeq Stranded mRNA Sample Preparation Guide (part # 15031047): The first step in the workflow involves purifying the poly-A containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM), followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The incorporation of dUTP in second strand synthesis quenches the second strand during amplification, because the polymerase used in the assay is not incorporated past this nucleotide. The addition of Actinomycin D to First Stand Synthesis Act D mix (FSA) prevents spurious DNA-dependent synthesis, while allowing RNA-dependent synthesis, improving strand specificity. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. The products are then purified and enriched with PCR to create the final cDNA library.  Unique Illumina TruSeq indexing adapter was ligated to this pool during adapter ligation step to be able to later pool several samples and pools in one flow cell lane. The high quality of the libraries was confirmed with Advanced Analytical Fragment Analyzer and the concentrations of the libraries were quantified with Qubit Fluorometric Quantitation, Life Technologies.  Only good quality libraries were sequenced. Typically the RNAseq library fragments are in the range of 200-700 bp and the average size of the fragments is 250-350 bp.