title: GSM8598197: Escherichia coli Nissle 1917, Tagatose condition, TEX untreated; Escherichia coli Nissle 1917; RNA-Seq
library_construction_protocol: The bacterial cultures were immediately mixed with two volumes of RNAprotect Bacteria Reagent (Qiagen) and incubated for 5 minutes at room temperature (RT). Following centrifugation at 10,000 g for 5 minutes at RT, the supernatant was discarded, and the cell pellets were stored at –80 °C if not processed immediately. Bacterial cells were lysed with 10 mg/ml lysozyme in TE buffer (pH 8.0) for 5 minutes at 25 °C. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. Residual DNA in the total RNA sample was digested with Turbo DNase (Applied Biosystems) for 30 minutes at 37 °C and removed using the filter cartridges provided with the mirVana kit. Purified RNA was stored at –80 °C. The quantity and purity of the purified RNA were assessed using a bioanalyzer (Agilent Technologies). High-quality RNA samples (RNA integrity number [RIN] > 8.0) were used for dRNA-seq library preparation. The dRNA-seq libraries were prepared as follows: half of the RNA was treated with terminator exonuclease (TEX, Epicentre), while equal amounts of untreated RNA were prepared as controls. For cDNA synthesis, RNA fragments were poly(A)-tailed using E. coli Poly(A) polymerase (NEB), and 5' triphosphate (5'PPP) structures were converted to 5' monophosphate (5'P) structures using RNA 5' Polyphosphatase (Epicentre). RNA sequencing adapters with barcodes were then ligated to the 5'-monophosphate ends of the fragments using T4 RNA ligase (NEB). Adapter-ligated RNAs were purified and size-filtered twice using 1.2× Agencourt AMPure XP beads (Beckman Coulter Genomics). First-strand cDNA was synthesized using an oligo(dT)-adapter primer and M-MLV reverse transcriptase (Invitrogen). The synthesized cDNA was purified using 0.8× Agencourt AMPure XP beads twice. cDNA amplification was carried out via PCR using Phusion High-Fidelity DNA Polymerase (NEB). The final amplified cDNA was purified using 0.8× Agencourt AMPure XP beads. Sequencing was performed on an Illumina NextSeq 500 system (Illumina) in 75 bp single-end mode.