title: Analysis of Host-Pathogen Interaction in Leaf Rust Infection of Wheat: A Transcriptomic Approach
design: The total RNA was isolated from susceptible (HD2329) mock-inoculated plants using TRI REAGENT (Molecular Research Center, Inc., USA). The RNA was treated with DNase I, amplification grade and RNaseOUT at 37°C for 30 min and quantified on a BioPhotometer (Eppendorf AG, Germany). Purified 25 µg total RNA was bound to 100 µl of Dynabeads Oligo(dT) EcoP magnetic beads by slowly rotating the tube on a rocking platform at 37°C for 30 min. The beads capture poly(A) RNA directly from total RNA. The first and second strand of cDNA was synthesized from the RNA on the beads using SuperScript III reverse transcriptase and E. coli DNA polymerase respectively. All the enzymatic steps were performed in one tube, to enhance the efficiency of cDNA synthesis. The double-stranded cDNA was digested with 10 µl of Nla III enzyme by incubating at 37°C  for 2 hrs. Gentle mixing was done by flicking the tube with finger every ~15 min. Nla III-digested cDNA was ligated to 1.5 µl adapter A using 2.5 µl of T4 DNA ligase (5U/ µl) at 16°C for 6 hrs. Gentle mixing was done by flicking the tube with finger every ~15 min. Adapter A ligated cDNA was digested with 10 µl of EcoP15I at 37°C for 6 hrs. Gentle mixing was done by flicking the tube with finger every ~15 min. EcoP15I digested cDNA i.e. the tags were ligated to 2 µl of adapter B using 2 µl of T4 DNA ligase (5U/ µl) at 16°C for 6 hrs. . Adapter B contains the other PCR priming site (P1) and SOLiD™ sequencing initiation sites. The quality of SAGE tags was determined by PCR using 0.5 µl of 5' (5’–CCACTACGCCTCCGCTTTCCTCTCTATG–3’) AMP primer and 3' (5’–CTGCCCCGGGTTCCTCATTCT–3’) AMP primer with 48 µl Platinum PCR SuperMix High Fidelity. After PCR amplification, the PCR products were run on 4% agarose gel with GeneRuler 50 bp DNA ladder (Fermentas GmbH, Germany). After analyzing the quality of the SAGE tags, process enters the SOLiD workflow.