GSM9610786: GC148685; Escherichia coli K-12; RNA-Seq
RNA was extracted using the NucleoSpin RNA kit (MACHEREY‑NAGEL, cat. 740955) following the manufacturer's instructions. Lysis was achieved by addition of buffer RA1 supplemented with β‑mercaptoethanol and the lysate was cleared using a NucleoSpin filter. 70% ethanol was added, followed by on‑column rDNase digestion, sequential washes and elution in RNase‑free water. RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies) with the Agilent RNA 6000 Nano Kit (Cat. No. 5067-1511) according to manufacturer's protocol. genomic DNA was extracted using a DNA purification kit according to manufacturer's instructions (Thermo Fisher Scientific), that yielded high-molecular-weight DNA suitable for next-generation sequencing library preparation Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (96 reactions; Cat. No. E7760L; Bioké). Ribosomal RNA was depleted using the NEBNext rRNA Depletion Kit v2 (Bacteria) with beads (24 reactions; Cat. No. E7860L; Bioké) to remove 16S/23S rRNA prior to cDNA synthesis. Standard steps included reverse transcription, second-strand synthesis, end repair, adapter ligation and PCR enrichment. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 in single-read mode using one lane of a non-shared flow cell.