title: GSM4773468: Cell aggregates of S. Typhimurium UMR1 [1_S1]; Salmonella enterica subsp. enterica serovar Typhimurium; RNA-Seq
library_construction_protocol: Cells were harvested by centrifugation and disrupted by lysis buffer (50 mM Tris HCL pH 8, 8 % sucrose, 0.5 % Triton, 20 mM EDTA, 4 mg/mL), 200 µL acidic phenol was added, and samples were then heated at 65 ºC for 15 min, while shaking. The sample was cooled on ice for 5 min and centrifuged at 13000 rpm. The aqueous phase was collected and extracted once with an acid phenol:chloroform mix, and twice with chloroform. Finally, RNA was precipitated with 3 volumes of 100 % ethanol and 0.3 M sodium acetate overnight at -80 ºC. Pellets were washed with 1 mL 70 % ethanol, and air-dried after the supernatant was removed. Pellet was dissolved in 100 µL DEPC-treated distilled water. After DNase (Ambion RiboPure-Yeast DNase) treatment, the RNA samples were analysed by denaturating gel electrophoresis to assess DNA contamination and integrity of the isolated RNA. If DNA contamination was still present, the RNA was repeatedly incubated with DNAse. The RNA concentration was measured by Nano drop and the quality of RNA preparation assessed by Agilent Bioanalyzer 2100. The DNA-free RNA was stored at -80 ºC until use. RNA samples were sequenced on a Nextseq 550 with medium output flowcell 150 cycles (2x75 PE) after depletion from ribosomal RNAs by the RiboMinus Transcriptome Isolation Kit (Invitrogen).