title: GSM9305516: E. coli lsrR deletion strain, mid-log phase (OD600=0.5), 3h, RNA-seq, replicate2; Escherichia coli K-12; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA integrity was confirmed using Agilent Bioanalyzer. Sequencing libraries were prepared using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's instructions. A total of 3 μg total RNA per sample was used as input. mRNA was enriched using poly-T oligo-attached magnetic beads. The purified RNA was fragmented under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H–) and random hexamers, followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H with dUTP substituted for dTTP to maintain directionality. After end repair and A-tailing, NEBNext adaptors with a hairpin loop were ligated. Size selection for ~150–200 bp fragments was performed using AMPure XP beads. The adaptor-ligated DNA was treated with USER enzyme at 37°C for 15 min and 95°C for 5 min. Libraries were PCR-amplified using Phusion High-Fidelity DNA Polymerase, universal primers, and index primers (X cycles according to kit recommendations). Final libraries were purified with AMPure XP beads and assessed using the Agilent 2100 Bioanalyzer. Library type: paired-end, directional (strand-specific).