Wild type Iron starvation 1h - Biological replicate 1
Wild-type (CEA17) and _ppzA (5x106 spores) were inoculated in MM for 24 hr at 37Ą and transferred to AMM (glucose 1%, trace elements without FeSO_) plus BPS 200 mM [Bathophenanthrolinedisulfonic acid (4,7-diphenyl-1,10 phenanthrolinedisulfonic acid)] and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine) 300 mM for 1 or 2 hr at 37Ą. Each condition was evaluated with three biological replicates. Mycelia were harvested, frozen in liquid nitrogen, and ground by mortar and pestle for total RNA isolation. 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 for the construction of the NGS libraries. Briefly, polyA containing mRNA molecules were selected using polyT oligo-attached magnetic beads. Fragmentation and 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 ÔAŐ nucleotide was added at the 3Ő end to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ÔTŐ nucleotide on the 3Ő 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.
SUB2558862
Sample_WT_1h_starv_I