Microbexpress kit

E. coli cell pellets were re-suspended in extraction buffer (10 mM Tris pH 8.0 and 1 mM EDTA) and incubated with 20 mg/ml lysozyme (Sigma) for 5 min at room temperature. The mixtures were then mixed in three volumes of TRIzol reagent (Invitrogen) and RNA was extracted by adding one volume of chloroform followed by centrifugation. Total RNA was precipitated in isopropanol and its quality and quantity were determined with a NanoDrop ND-1000 spectrophotometer (Thermo) and TAE agarose gel electrophoresis.
Small RNA (sRNA) was separated and enriched from total RNA by utilizing the mirVana™ miRNA Isolation Kit (Life Technologies) and subjected to the MICROBExpress Kit (Ambion) and Ribo-Zero rRNA Removal Kit (Epicenter) to eliminate rRNA according to the manufacturer's instructions. The concentration of sRNAs was also confirmed by ND-1000 (Thermo), and its quality and integrity were then monitored by Bioanalyzer (Agilent) using RNA 6000 Pico Kit (Life Technologies).

The wild-type, ΔrhpS, and ΔrhpRS strains were cultured in KB medium until they reached an OD₆₀₀ of 0.6 before being transferred to liquid MM for 6 h. Then, 2 ml of bacterial cultures was collected by centrifugation (12,000 rpm at 4°C). RNA purification was conducted with an RNeasy minikit (Qiagen). After removal of rRNA by using the MICROBExpress kit (Ambion), mRNA was used to generate the cDNA library according to the NEBNext UltraTM II RNA Library Prep kit protocol (NEB), which was then sequenced using the HiSeq 2000 system (Illumina). Bacterial RNA-seq reads were mapped to the Pseudomonas savastanoi pv. phaseolicola 1448A genome (NC_005773.3) by using STAR. Only the uniquely mapped reads were kept for the subsequent analyses. The gene differential expression analysis was performed using Cuffdiff software (version 2.0.0) (64 (link)). GO enrichment analyses were conducted on all differentially transcribed genes using DAVID (65 (link)). Each sample analysis was repeated twice.

Total RNA was purified from frozen bacteria pellets through trizol extraction using 1 ml of the reagent during 5 min at room temperature. Membrane debris were removed with a 15 min centrifugation at 13000 g at 4 °C. All reagents were gently mixed and incubated for 5 min at room temperature before centrifugation. All subsequent centrifugations lasted 5 min. After centrifugation with 400 μl of chloroform/isoamyl alcohol (24/1), the aqueous phase was treated with 500 μl acidic phenol and an additional 300 μl chloroform/isoamyl alcohol mix. Total RNA was then washed a second time with 200 μl chloroform/isoamyl alcohol mix, and then precipitated with 250 μl isopropanol for 15 min on ice. Total RNA was later centrifuged 15 min at 13000 g at 4 °C; pellets were washed with 1 ml 70% ethanol and centrifuged 5 min at 13000 g at 4 °C. Pellets were then air dried and resuspended for 15 min at 60 °C in 200 μl Tris 10 mM EDTA 0.1 mM pH 7.4. Genomic DNA was removed using 2 μl Turbo DNase (AMBION) for 30 min at 37 °C. Total RNA was quantified using Thermo Fisher’s NanoDrop. RNA integrity was verified on a Nano6000 RNA chip using the Bioanalyzer 2100 (Agilent). mRNA enrichment was performed with the MICROBExpress Kit (Ambion) from 7 μg of total RNA per sample. Depletion of 16S and 23S ribosomal RNAs was confirmed on a Nano6000 RNA chip with the Bioanalyzer 2100 (Agilent).