title: RNA-Seq of A. fumigatus wild-type strain in the presence of calcoflour white (replicate 1)
design: Total RNA was extracted using Trizol (Invitrogen), treated with RNase-free DNase I (Qiagen) and purified using a RNAeasy Kit (Qiagen) according to manufacturer's instructions. RNA from each treatment was quantified using a Qubit fluorometer and analyzed using an Agilent 2100 Bioanalyzer system to assess the integrity of the RNA. Illumina TruSeq Stranded mRNA Sample Preparation kit was used to construct cDNA libraries following manufacturer's instructions. Briefly, polyA containing mRNA molecules were selected using polyT oligo-attached magnetic beads. Fragmentation and library preparation were done using divalent cations and thermal fragmentation. First strand cDNA synthesis was performed using reverse transcriptase (Superscript II) and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H and dUTP in place of dTTP. AMPure XP beads were used to separate the dscDNA from the second strand. At the end of this process, we had blunt-ended cDNA to which a single A nucleotide was added at the 3 end to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single T nucleotide on the 3 end of the adapter provided a complementary overhang for ligating the adapter to the fragment. The products were then purified and enriched using a PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The PCR was performed with a PCR Primer Cocktail from the Illumina kit that anneals to the ends of the adapters.