RNA-Seq of Ascochyta rabiei in PDB Read 2
Total RNA was isolated from mycelium of A. rabiei isolate P4 growing for one week in PDB shaking media containing ampicillin and kanamycin (50mg/ml each) in darkness at 20ą2 şC. Two independent replicates were performed.RNA was extracted using TRISure.  Potential residual genomic DNA was removed using RQ1 RNase-Free. RNA was further purified using the RNeasy Plant Mini Kit. RNA-seq libraries were generated from 5?g of total RNA by first capturing the mRNA using Oligo dT(25) beads. The purified mRNA was then randomly fragmented in a Zn2+ solution and Illumina sequencing adapters were ligated to the RNA. cDNA was then generated using a p5 oligonucleotide for priming and the library was finally amplified with 10 cycles of PCR. To avoid any possible contaminant or artifacts, the sequences were mapped against the A. rabiei genome and only those that mapped were retained