GSM5830894: ∆hmpX1; Ralstonia solanacearum; RNA-Seq
GSM5830894
After 16h total incubation, sub-samples were collected for dilution plating to determine CFU/ml and the tubes were capped and centrifuged at room temperature for 5 min at 8000 rpm. The supernatant was removed and pellets were frozen in liquid nitrogen. RNA extractions were carried out using a modified version of the Quick-RNATM MiniPrep kit (Zymo Research, Irvine, CA, USA), as follows. Pellets were resuspended in 400 uL of ice cold TE pH 8 with 1 mg/mL lysozyme, 0.25 uL Superase Inhibitor (Ambion, Austin, TX, USA), and 80 uL of 10% SDS, vortexed for 10s, transferred to a new 2 mL tube, and shaken at ~300 rpm for 2 min. 800 uL of RNA-Lysis lysis buffer was added, the tubes were vortexed again for 10s, cleaned according to the kit manufacturer's instructions, and eluted in 100 uL of water. Nucleic acid concentrations were estimated using a nanodrop, normalized to 200 ng/uL, and cleaned using the DNA-free DNAse kit (Invitrogen, Carlsbad, CA, USA). Samples were incubated at 37C for 1 hour, with 2 uL more of DNAse added at 30 min. After DNAse inactivation, samples were further cleaned by chloroform extraction, then precipitated overnight at -20C with 100 microM Sodium Acetate pH 5.5 and 66 % ethanol. Samples were checked for concentration on a Nanodrop, for DNA contamination by PCR using the qRT-PCR primers serC_F/R, and for RNA integrity (RIN) using an Agilent Bioanalyzer 21000 (Agilent, Santa Clara, CA, USA). All samples had RIN values above 7.3. NEBNext uLtraTM Directional RNA Library Prep Kit for Illumina (NEB, Ipswitch, MA, USA) following the manufacturer's recommendation and starting with 3 mg of RNA per sample