title: GSM8035282: RNA S1K247Q-3; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
library_construction_protocol: Bacterial samples were treated with pre-chilled cell lysis buffer [1% Triton X-100 in ribosome buffer (RB buffer): 20 mM HEPES-KOH (pH 7.4), 15 mM MgCl2, 200 mM KCl, 100 𝜇g/ml cycloheximide, and 2 mM dithiothreitol]. After a 30 min ice bath, cell lysates were collected and transferred to pre-chilled 1.5 mL tubes. After that, cell debris was removed by centrifuging at 4 °C for 10 min. Supernatants were transferred to the surface of sucrose buffer (30% sucrose in ribosome buffer), then ultracentrifuged at 4°C by T-865 fixed angle rotor (ThermoFisher, Germany) to pellet RNCs. Pellets were then resuspended with RB buffer. Isolation of total RNA was carried out by the Trizol Reagent (Invitrogen, USA) according to the manufacturer's instructions. Sequencing libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA, Catalog #: E7530L) following the manufacturer's instructions.