RNA-seq of BRA: Resistant bulk replicate 1
The BC1 population was employed for a BSR-seq analysis. At 15 days post-inoculation, an R bulk and an S bulk were formed by combining leaf tissue from 30 resistant and susceptible BC1 plants, respectively. One biological replicate was constituted by these two bulks, and three biological replicates were used for RNA sequencing. The RNeasy Plant Mini Kit (Qiagen; Toronto, Canada) was utilized for total RNA extraction from each bulk, with on-column DNAse digestion performed using a Qiagen RNase-Free DNase Set following the manufacturers instructions. Assessment of RNA concentration and purity was carried out using a NanoDrop ND-2000c spectrophotometer. The Experion RNA StdSens analysis kit (Bio-Rad; Montreal, Canada) was employed to ensure RNA quality, with a stipulation that the RNA integrity number (RIN) should exceed 8 for each sample. Subsequent steps in the process included cDNA library preparation following the TruSeq RNA Sample Preparation v2 Guide (Illumina; San Diego, USA). The NanoDrop ND-2000c spectrophotometer was utilized to verify cDNA concentrations and purity. Validation of the cDNA libraries through quality control and qPCR analysis was conducted, and the Experion DNA 1 K Analysis Kit (Bio-Rad) was utilized to confirm their size and purity based on a band at approximately 260 bps. Lastly, RNA-Seq was performed on samples from each inoculated R and S bulk utilizing the Illumina MiSeq platform at the University of Saskatchewan (Saskatoon, Canada).