RNAseq of Syzygium luehmannii leaf:inoculated with Puccinia psidii
RNA samples (24 samples of ~500 ľg each) were prepared for mRNA sequencing using the NEBNextŽ Ultra RNA Library Prep Kit for IlluminaŽ (#E7530) (Illumina Inc. San Diego, CA, USA) according to the manufacturers protocol. Eight sample libraries were redone, due to initial low concentrations, with NEBNextŽ Ultra Directional RNA Library Prep Kit for IlluminaŽ (#E7420).Size selection from fragment lengths of 300-450 bp insert size was based on the recommendations in Appendix A, Chapter 4 of the protocol. Specifically, during Chapter 1.2, step 37, the samples were incubated at 94?C for 10 minutes and during Chapter 1.3, First Strand cDNA synthesis  incubation at 42?C was extended from 15 minutes to 50 minutes. For Chapter 1.8, Size Selection of Adaptor-Ligated DNA, we referred to Chapter 4, Appendix A and used 45 ľL of magnetic beads at step 3. At Chapter 1.9A, step 2, I used 14 cycles of denaturation, annealing/extension. Library concentrations were assessed using Qubit Fluorometric Quantitation and samples diluted to ~ 3 nM concentrations before pooling. All bead clean-ups were conducted with GE Healthcare Life Sciences Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (hydrophobic) 15 mL (GE Healthcare Life Sciences, Chicago, IL, USA) except for the initial steps, Chapter 1.2, using NEBNext Oligo d(T)25. Sequencing was run using two lanes of an Illumina HiSeq2500 (Illumina Inc. San Diego, CA, USA) at the Australian National University Biomolecular Resource Facility with 150 bp Paired End reads