title: Aspergillus fumigatus Wild Type 0min 1M Sorbitol - Replicate II
design: A. fumigatus conidia (1x106 sp/mL) were inoculated into liquid YPD (1% yeast extract, 1% polypeptone, and 1% glucose) and cultured for 16 h prior to addition of 1/2 vol. 3 M sorbitol (Final concentration: 1 M). Mycelia were harvested, frozen in liquid nitrogen. For total RNA isolation, mycelia were ground in liquid nitrogen. Total RNA was extracted using Trizol (Invitrogen), treated with RNase-free DNase I (Fermentas) and purified using a RNAeasy Kit (Qiagen) according to manufacturer's instructions. The RNA from each treatment was quantified using a NanoDrop and Qubit fluorometer, and analyzed using an Agilent 2100 Bioanalyzer system to assess the integrity of the RNA. RNA Integrity Number (RIN) was calculated; RNA sample had a RIN = 9.0 - 9.5.  Illumina TruSeq Stranded mRNA Sample Preparation kit was used. Briefly, polyA containing mRNA molecules were selected using polyT oligo-attached magnetic beads. Fragmentation and paired-end library preparation was 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. Libraries were sequenced (2x100bp) on a HiSeq 2500 instrument, sequencing approx. 17.3x106 fragments per sample.  Analysed samples:    WT 0min exposure to 1M sorbitol - 3 biological replicates  WT 10min exposure to 1M sorbitol - 3 biological replicates  DsakA 0min exposure to 1M sorbitol - 3 biological replicates  DsakA 10min exposure to 1M sorbitol - 3 biological replicates  DmpkC 0min exposure to 1M sorbitol - 3 biological replicates  DmpkC 10min exposure to 1M sorbitol - 3 biological replicates  DmpkCDsakA 0min exposure to 1M sorbitol - 3 biological replicates  DmpkCDsakA 10min exposure to 1M sorbitol - 3 biological replicates