Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: 20 ml culture were collected by vacuum filtration through 0.8 µm polyethersulphone filter disks (Pall, Germany). Filters were immediately transferred to 1.6 ml PGTX solution (Pinto et al., 2009), vortexed, frozen in liquid nitrogen and stored at -80°C. RNA was extracted according to Pinto et al. (2009) with modifications as described in Wallner et al. (2020). For RNA Sequencing, biological triplicates of WT and rne(WT) as well as biological duplicates of rne(5p) were used. Residual DNA was removed from samples containing each 10 µg RNA by three subsequent incubation steps with Ambion TURBO DNase (Thermo Fisher Scientific, Germany). For each step, 2 U TurboDNase was added, followed by 20 min incubation at 37°C. RNA was recovered using RNA Clean & Concentrator kits (Zymo Research, USA). RNA integrity was controlled for on a Fragment Analyzer using the High Sensitivity RNA Analysis Kit (Advanced Analytical Technologies, Germany). cDNA libraries were constructed and sequenced as a service provided by vertis Biotechnologie AG (Germany) according to the tagRNA-Seq protocol (Innocenti et al., 2015). Ribosomal RNAs were depleted using in-house depletion probes. 5'-P RNA fragments were ligated to the 5' Illumina TruSeq sequencing adapter carrying the sequence tag CTGAAGCT. After incubation with 5'-phosphate-dependent exoribonuclease Terminator Exonuclease (TEX, Lucigen), samples were treated with RNA 5' Polyphosphatase (5'PP, Lucigen). The 5' Illumina TruSeq sequencing adapter carrying sequence tag TAATGCGC was ligated to newly formed 5'-P RNA ends. RNA was fragmented and an oligonucleotide adapter was ligated to the resulting 3' ends. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. First-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. This was followed by PCR amplification to about 10-20 ng/µl using a high fidelity DNA polymerase. cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). Samples were pooled in approximately equimolar amounts and the cDNA pool in the size range of 200 – 600 bp was eluted from a preparative agarose gel.