Xanthomonas citri subsp. citri 306; widltype; overexpressing tagK in pBRA vector; mid-log phase; biological replicate 1
Xanthomonas citri subsp. citri 306 overexpressing tagK in pBRA vector (WT/tagK++) was cultured at 28C with shaking in LB medium until mid-logarithmic phase (OD600nm 0.5). 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 65C 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). Libraries were prepared with TruSeq Stranded mRNA Kit (Illumina) using the Low Throughput (LT) protocol, as recommended by the 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 three replicates were obtained for each strain and this sample corresponds to the biological replicate one for WT/tagK++. The following sequences may be used to trim adapters. Read 1: AGATCGGAAGAGCACACGTCTGAACTCCAGTC; Read 2: AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT.