2.2. RNA Extraction, Library Preparation, and High-Throughput Sequencing

RNA Extraction, library preparation, and high-throughput sequencing were performed as previously described by Veselovsky et al. [16]. Total RNA extraction and purification was performed using the RNeasy Mini Kit (QIAGEN). Removal of residual gDNA was performed using the TURBO DNA-free Kit (Invitrogen, Waltham, MA, USA) and the RNase-Free DNase Set (QIAGEN, Hilden, Germany). The concentration and quality of the extracted RNA were checked using the Quant-iT RiboGreen RNA Assay Kit (Thermo Fisher Scientific, Walthamm, MA, USA) and the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA), respectively.

Total RNA (2 µg) was used for library preparation. Ribosomal RNA was removed from the total RNA using the RiboZero rRNA Removal Kit (Bacteria) (Epicentre/Illumina, Madison, WI, USA), and libraries were prepared using the NEBNext^® Ultra II Directional RNA Library Prep Kit (NEB, Ipswich, MA, USA), according to the manufacturer protocol. Subsequently, RNA cleanup was performed with the RNA°Clean XP kit (Beckman Coulter, Brea, CA, USA). The library underwent a final cleanup using the Agencourt AMPure XP system (Beckman Coulter, Brea, CA, USA) after which the libraries’ size distribution and quality were assessed using a high sensitivity DNA chip (Agilent Technologies). Libraries were subsequently quantified by Quant-iT DNA Assay Kit, High Sensitivity (Thermo Fisher Scientific). Finally, equimolar quantities of all libraries (12 pM) were sequenced by a high throughput run on the Illumina HiSeq using 2 × 100 bp paired-end reads and a 5% Phix spike-in control. Before loading the cBot system, the libraries were incubated at 98 °C for 2 min and then cooled on ice to improve the hybridization of GC-rich sequences. The dataset of RNA-Seq analysis was deposited to the NCBI under the project name PRJNA628664.