Total RNA was extracted using the RNAprep pure Plant Kit (Tiangen) with on-column DNaseI digestion from samples of 0, 1, 4 dpi. Reverse transcription was conducted with equal amount of total RNA (5 μg) using the SuperScript® III First-Strand Synthesis System (Invitrogen), and the resulting cDNA was diluted 1:3 with nuclease-free water.
Each PCR reaction used 3 μL cDNA. The PCR amplification was conducted using the Hot Start TaqDNA polymerase (Takara) in 50 μL volume. The following cycling condition was used: 95°C for 10 min and 38 cycles at 95°C for 15 s, 56°C for 30s and 72°C for 1 min. PCR products of IGYP genes in different infection time points were pooled separately, and 1 mL of pooling PCR products were gel-purified to remove >400-bp products and primer dimers and eluted by 200 μL ddH2O.
The purified samples were used to build libraries using a TruSeq DNA sample prep kit (Illumina). The TruSeq libraries were sequenced using Illumina HiSeq2000 with 100-bp paired-end reads. Each library produced more than 2 Gb raw data. Low-quality bases (≦Q20), adaptors and short reads (≦50 bp) were removed. The sequences were aligned using TopHat2 software against the gDNA reference sequences of IGYPs[42 (link)].
Each PCR reaction used 3 μL cDNA. The PCR amplification was conducted using the Hot Start TaqDNA polymerase (Takara) in 50 μL volume. The following cycling condition was used: 95°C for 10 min and 38 cycles at 95°C for 15 s, 56°C for 30s and 72°C for 1 min. PCR products of IGYP genes in different infection time points were pooled separately, and 1 mL of pooling PCR products were gel-purified to remove >400-bp products and primer dimers and eluted by 200 μL ddH2O.
The purified samples were used to build libraries using a TruSeq DNA sample prep kit (Illumina). The TruSeq libraries were sequenced using Illumina HiSeq2000 with 100-bp paired-end reads. Each library produced more than 2 Gb raw data. Low-quality bases (≦Q20), adaptors and short reads (≦50 bp) were removed. The sequences were aligned using TopHat2 software against the gDNA reference sequences of IGYPs[42 (link)].
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