GSM8494190: Untreated control 10 min post-treatment; BR 2; Escherichia coli; RNA-Seq
Transcriptomics was sampled according to the protocol provided with RNeasy Mini kit (Qiagen). The sample volume was based on OD600 at each sampling timepoint to ensure 3.35 x 10^8 cells per sample. The sample was immediately added to 2 volumes of RNA protect and vortexed. After minimum 5 min, the samples were centrifuged (10 min, max rcf). The supernatant was discarded, and the pellet was frozen in N2 (l). RNA was isolated using RNeasy Mini Kit (Qiagen), according to manufacturer's protocol 4 (Enzymatic lysis and Proteinase K digestion of bacteria) and protocol 7 (Purification of total RNA) with on-column Dnase (Qiagen) digestion. The RNA concentration in each sample was measured using NanoDrop-1000 spectrophotometer. Isolated RNA was sent to the Genomics Core Facility at NTNU for further processing and sequencing. RNA sequencing libraries were prepared using the QIAseq FastSelect 5S/16S/23S kit (Qiagen) for rRNA removal and the QIAseq stranded RNA Lib kit (Qiagen) for library construction, according to the manufacturer's instructions.  Briefly, 500 ng total RNA was used as starting material. Removal of ribosomal RNA (rRNA) was conducted by a combined heat fragmentation (89°C for 7 min) and FastSelect hybridization protocol (75-4°C ramping process) where the FastSelect reagent inhibited reverse transcription of bacterial rRNA. Next, purification was conducted using QIAseq Beads followed by a first-strand synthesis using a RNase H- Reverse Transcriptase (RT) in combination with random primers, a second-strand synthesis, end-repair, A-addition, and adapter ligation. The second-strand synthesis was performed using 5'phosphorylated random primers which enable subsequent strand-specific ligation. DNA fragments were further enriched by CleanStart library amplification (15 cycles of PCR reaction). Finally, the libraries were purified using the QIAseq Beads, quantitated by qPCR using Collibri Library Quantification Kit (Thermo Fisher Scientific), and validated using Perkin Elmer DNA 1K/12K/Hi Sensitivity Assay LabChip on a Labchip GX instrument (Perkin Elmer, USA). The size range of the DNA fragments were measured to be in the range of 270 to 570 bp and peaked around 355 bp. Prior to sequencing, libraries were normalized and pooled to 2.3 pM and subjected to clustering on three NextSeq 500 HO flowcells (Illumina, USA).