title: RNA seq of Triticum aestivium: shoot base of seedlings: Sample 9B_fusarium_5dpi
design: "Five sets of near isogenic lines (NILs) for the FCR QTL on chromosome arm 3BL reported were used in this study. They included'1A/1B','2A/2B','3A/3B','4A/4B', and'9A/9B'.'A'isolines are those carrying the resistant allele and'B'isolines are those carrying the susceptible allele at the Qcrs-3B locus. Ten seedlings were used in each of the biological replications for all of the experiments conducted. Two-day-old seedlings were inoculated with the Fusarium. pseudograminearum isolate (Fp-infection) or water (mock). Samples were taken by cutting the shoot base (0-4 cm) at 3 or 5 days post inoculation (dpi) and frozen in liquid nitrogen immediately and kept at -80 degree C until processed. Two datasets of RNA sequences were obtained in this study. The first dataset was obtained from the'1A/1B'NIL set. The experimental design for the'1A/1B'contained two treatments (mock and Fp-infection), two time points (3 and 5 dpi) and six biological replicates. The second one was generated from the other four sets of NILs, which were used only to validate those differentially expressed genes with SNPs (SNP-EGs) detected from the'1A/1B'NIL set. A single trial with three biological replications was conducted for these four sets of NILs. Mocks were not used and samples were collected at 5 dpi only. Before RNA isolation, samples from the three biological replications for each of the eight isolines were pooled. Thus, a total of 8 samples (each for a different line of the four sets of NILs) were used for RNA extraction. Total RNA was isolated using a QIAGEN RNeasy plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, using RLT buffer and including the optional on-column DNase I digestion. The yield and purity of each RNA sample was determined by the absorbance (Abs) at 260 and 280 nm and the integrity of all RNA samples was assessed on 1 % agarose gels. Each sample of 10 micro g of total RNA was sent to Australian Genome Research Facility Ltd (Parkville, Victoria, Australia) for further processing before Illumina HiSeq 2000 sequencing. Two technical replications were run for each of the 56 (48 for '1R/1S'and 8 for the other 4 sets of NILs) cDNA libraries with 6 lanes of 100 bp paired-end sequencing."