Genemapper software v

5 μg of total cellular RNA of N44-transfected cells was reverse transcribed with the First-Strand cDNA Synthesis Kit (ThermoScript RT-PCR system). The 6-FAM labeled primer (5′-GATATCACCGGTATATTA-3′) complementary to the 3′ end of the N44 RNA sequence was used for primer extension. After incubation with RNase H, the cDNA was concentrated by ethanol precipitation and mixed with 1.5 μl Rox 500 Size Standard and run on an ABI PRISM 3010 XL Genetic Analyzer (Applied Biosystems) using default parameters. All data were analyzed using GeneMapper® software v4.0 (Applied Biosystems) and then manually corrected based on fragment size to generate the N44 transcription start site profile.

The touchdown PCR profile was used for screening of LLS and rust-resistant alleles using linked markers. The reaction mixture consisted of 5 ng of DNA, 2 pmol of M13 labeled forward primer, 5 pmol of reverse primer, 2 mM MgCl₂, 2 mM dNTPs, 0.1 U of Taq DNA polymerase (Kappa Taq) and 1X PCR buffer. A standardized touch down PCR program with 5 min initial denaturation, followed by 5 cycles of 94°C for 20 s, 65°C for 20 s and 72°C for 30 s with 1°C decrement for every cycle, followed by 40 cycles of 94°C for 20 s, constant annealing temperature of 59°C and 72°C for 30 s ending with extension for 20 min at 72°C. The PCR products were resolved on 2% agarose gel for confirmation of amplification. In SSRs, forward primers were dye-labeled with FAM, VIC and NED which were detected as blue, green and black color peaks, respectively, upon capillary electrophoresis. The PCR products were denatured and capillary separated with ABI 3700 automatic DNA sequencer (Applied Biosystems, United States) and GeneMapper Software V (Applied Biosystems) was used to analyze the peak patterns.

The resulting fluorescently-labeled cDNA fragments were analyzed in a 3730 capillary sequencer (Applied Biosystems) and the sequencing files were visualized with the GeneMapper software v.4.0 (Applied Biosystems). To accurately assign a nucleotide base to in vitro transcription or primer extension peaks, a sequencing ladder was generated using the above lctP template DNA, primer HEX-lctP-IvT and the Thermo Sequenase Dye Primer Manual cycle sequencing kit (USB Corporation) as described before [65 (link)]. The GeneMapper software was used to generate alignments between the electropherograms of the transcription reactions and dideoxy sequencing reactions to determine the size and start nucleotide of the lctP transcripts. The effect of GdhR and MtrR on lctP in vitro transcription was estimated from the height of lctP transcript peaks present in the electropherograms. Transcription inhibition curves were generated with the aid of GraphPad Prism 5.0 (GraphPad, San Diego, CA) using the log (inhibitor) vs. response-variable slope nonlinear regression analysis.