title: CJS_3
organism: Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819
description: Protocols: Bacterial cultures (12 ml BHI) of C. jejuni strains were grown under microaerobic conditions to OD600 of 0.5-0.7. Immediately after opening the jar, 2 ml of the non-stressed culture was added to 2 ml of the RNAprotect Bacteria Reagent (Qiagen), vortexed and incubated for 5 min at room temperature. In parallel, the cultures were treated with 1 mM paraquate (final concentration) for 25 min (42 °C, 140 rpm orbital shaking). After oxidative stress, samples were collected similarly to non-stressed cells. After 5 min incubation with RNAprotect Bacteria Reagent, bacteria were collected by centrifugation (4.700 × g, 10 min, RT). RNA was isolated by GeneJET RNA Purification Kit (Thermo Fisher Scientific; K0731) according to the manufacturer's protocol and treated with RNase-free DNase I (Thermo Fisher Scientific). Next, the subsequent purification by the GeneJET RNA Purification Kit was performed to remove DNase I. A NanoDrop Lite spectrophotometer, agarose gel electrophoresis and Agilent 4200 TapeStation System were used to determine the RNA quality and quantity. RNA was isolated immediately after bacteria collection, stored at -80 °C for up to one month and used for RNA sequencing. RNA was isolated from three independent cultures (three biological replications). The prokaryotic directional mRNA library preparation was performed at the Novogene Bioinformatics Technology Co. Ltd. (Cambridge, UK). The ribosomal RNA was removed from the total RNA, followed by ethanol precipitation. After fragmentation, the first strand of cDNA was synthesised using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit, RT-qPCR for quantification and a bioanalyser for size distribution detection.