GSM5738301: Expo_Rep3_A; Escherichia coli; RNA-Seq
Total RNA was extracted using the RNeasy Midi kit (Qiagen, Germantown, MD, USA) and quantified using a Nanodrop® spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The 23S/16S and 16S/23S ratios in each subfraction were calculated using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and used to pool the subfractions into seven fractions labelled A to G. Fraction A consisted of subfractions containing DNAs, free RNAs, and free small and large ribosomal subunits. In fractions B to G, the 23S/16S ratio was constant, at about 1.8, corresponding to entire ribosomes. The 1st peak (fraction B) was attributed to the monosome. The 2nd, 3rd, and 4th peaks were attributed to 2, 3, and 4 ribosomes, respectively. Ribosomal RNA was removed using the Ribo-ZeroRM Magnetic Kit (Illumina, San Diego, CA, USA). Then 10 ng ribodepleted RNA was used to construct a sequencing library using the Ion Total RNA-Seq Kit v2 Kit and the Ion Xpress™ RNA-Seq Barcode 1-16 Kit (Thermo Fisher Scientific, Waltham, MA, USA). The library was constructed and (100 base pair [bp]) single-end sequencing on an Ion Torrent system equipped with an Ion P1 Chip Kit was performed by the GeT-BioPuces platform (http://get-biopuces.insa-toulouse.fr/). These experiments were repeated with three independent cultures.