Xanthomonas citri subsp. citri 306; widltype; overexpressing ecfL in pBRA vector; mid-log phase; biological replicate B2
Xanthomonas citri subsp. citri 306 overexpressing ecfL in pBRA vector (WT/ecfL++) was cultured at 28 C with shaking in 2xTY medium until mid-logarithmic phase (OD600nm 0.6). For RNA extraction, cells from a 6 ml sample were harvested by centrifugation at 16 000 g for 1 minute and lysed with 1ml Trizol (Ambion), followed by incubation for 10 minutes at 65 C and extraction with 0,2 volumes of chloroform. The aqueous phase was purified with the RiboPure Bacteria kit (Ambion). DNA was removed using TURBO DNA-free kit (Ambion) and sample quality was analyzed by running Agilent RNA 6000 Pico Kit using Agilent 2100 Bioanalyzer instrument. Samples were treated with Ribo-Zero rRNA Removal Kit (Illumina) and a second analysis was performed with Agilent RNA 6000 Pico Kit and Agilent 2100 Bioanalyzer instrument. Libraries were prepared with TruSeq RNA Sample Prep Kit v2 (Illumina) using the Low Throughput (LT) protocol, as recommended by manufacturer. Quality control was performed by running Agilent DNA 1000 kit using Agilent 2100 Bioanalyzer. Libraries were quantified by qPCR using KAPA Library Quantification Kit Illumina platforms (KAPA Biosystems) and normalized to 4 nM. Sequencing was performed using the NextSeq 500/550 Mid Output v2 kit (150 cycles, 2x75) and the NextSeq instrument, at the Genial Core facility (Genome Investigation and Analysis Laboratory) / CEFAP (Centro de Facilidades para a Pesquisa) - University of Sao Paulo (USP). A total of 2 biological replicates were obtained for each strain and this sample corresponds to the biological replicate one (B2) for WT/ecfL++. The following sequences may be used to trim adapters: Read 1: AGATCGGAAGAGCACACGTCTGAACTCCAGTCA; Read 2: AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT