title: Illumina HiSeq 2000 paired end sequencing; Transcriptome analysis in the old Brazilian wheat variety Toropi in response to infection with Puccinia triticina, the causal pathogen of leaf rust
design: Transcriptome analysis in the old Brazilian wheat variety Toropi in response to infection with Puccinia triticina, the causal pathogen of leaf rust
library_construction_protocol: The flag leaf on the first tiller of each plant was inoculated with urediniospores of Pt race MDT-MR suspended in mineral oil, or mineral oil alone (Mock-inoculation). Pt- and Mock-inoculated plants were collected at 0, 6, 12 and 24 hours after inoculation (hai), the leaf tissue being placed immediately into liquid nitrogen.  Individual plants of the Brazilian wheat cv. Toropi (seed obtained from Embrapa-Wheat, Brazil) were grown to first flag leaf emergence under a 14 h light/20 ºC and 10 h dark/16 ºC growing cycle, at 80% humidity. Each plant was grown in a soil substrate (rice husk, peat and humus) and vermiculite mix in a 1 liter pot.  The flag leaf on the first tiller of each plant was inoculated with urediniospores of Pt race MDT-MR suspended in mineral oil, or mineral oil alone (Mock-inoculation). Pt- and Mock-inoculated plants were collected at 0, 6, 12 and 24 hours after inoculation (hai), the leaf tissue being placed immediately into liquid nitrogen.  RNA was extracted from flag leaves using RNAeasy Plant Mini kit (Qiagen) and purified using the Turbo DNA-freeTM kit (Invitrogen).  For each time point (0, 6, 12, 24 hai) RNA from three Pt and three Mock-inoculated flag leaves was sent to the Earlham Institute (formerly The Genome Analyses Centre), Norwich, UK for library construction and paired-end sequencing.