title: GSM7112576: 52-S1 1; Pseudomonas fluorescens; RNA-Seq
library_construction_protocol: Whole-cell RNA was extracted from 20 OD units of P. fluorescens cultures in mid-log phase growth (OD595 ~1.5). Upon reaching the desired OD, growth and RNA expression were halted by addition of a ½ culture volume of ice-cold killing buffer (20 mM NaN3, 20 mM Tris-HCl, 5 mM MgCl2). Cells were pelleted, and the killing buffer removed. A lysis buffer of β-mercaptoethanol in buffer RLT from the Qiagen RNeasy extraction kit was used to resuspend pellets, which were then lysed by bead-milling at 4500 rpm for 45 s with lysing matrix B. Lysates were spun through columns from the Qiagen RNeasy extraction kit, and the extraction completed following the RNeasy kit protocol. A DNase I treatment step was included between washes with buffer RW1, by adding DNase I directly to the column from the RNase-free DNase kit (Qiagen) following kit protocol. Samples were eluted in nuclease-free water, and subsequently treated with TURBO DNase from the Turbo DNA-free kit (Invitrogen) following kit protocols. RNA sequencing was provided by Oxford Genomics (Oxford UK), and followed their methods for library preparation and sequencing. Samples were ribodepleted, and the mRNA fraction converted to cDNA with dUTP incorporated during second strand synthesis. The cDNA was end-repaired, A-tailed and adapter-ligated. Prior to amplification, samples underwent uridine digestion. The prepared libraries were size selected, multiplexed and QC'ed before paired end sequencing over one unit of a flow cell in a NovaSeq 6000.