title: GSM8949908: S. Typhi treated with 0.1% DMSO. Exp3.; Salmonella enterica subsp. enterica serovar Typhi; RNA-Seq
library_name: GSM8949908
library_construction_protocol: S. Typhi was grown in LB overnight and 50 µl of culture was inoculated into 2 ml of 86 mM NaCl-LB containing 0.1% DMSO (control) or 2 µg/ml rifampin. When OD600nm reached 0.9-1.0, 1.5 ml of the culture was pelleted at 8,000 ×g for 2 min; the pellets were immediately resuspended in 1 ml of RNAprotect Bacteria Reagent (Qiagen, cat# 76506) and incubated for 10 min at room temperature. The bacterial pellets were stored at -80°C overnight. On the following day, total RNAs were isolated using RNeasy mini kit (Qiagen, cat# 74104) with in-column DNase digestion (Qiagen, cat# 79254). RNA quantity and quality were evaluated using NanoDrop 2000 spectrophotometer (ThermoFisher), Qubit 4 fluorometer (ThermoFisher), and standard RNA gel electrophoresis. The following two-step bacterial rRNA depletion procedures were performed to obtain high-quality mRNA samples. First, 5 µg total RNA was treated with RNASEOUT (Invitrogen, cat# 10777019) and Terminator 5'-Phosphate-Dependent Exonuclease (Lucigen, cat# TER51020) for 1 hr at 37°C. Second, the remaining rRNA in the sample was further depleted with NEBNext® rRNA Depletion Kit for bacteria (NEB cat# E7850L). After rRNA depletion, the random-primed double-stranded cDNA (dscDNA) library was prepared with the Maxima H Minus Double-Stranded cDNA Synthesis Kit (ThermoFisher, cat# K2561). The purified dscDNA library was end-repaired (NEB, #cat E7546L) and barcoded (ONT, #cat EXP-NBD104). All the barcoded samples were pooled for adaptor ligation (AMII, part of ONT EXP-NBD104). RNAclean XP beads (Beckman Coulter, #cat A63987) and AMPure XP beads (Beckman Coulter, cat # A63880) were used at each step that requires RNA and DNA purification, respectively.
identifiers.namespace: GEO
identifiers.id: GSM8949908_r1