Microbe express kit

The mid-exponential growth phase was chosen for RNA sequencing, corresponding to 4.5 hpi for BP and CT and to 6 hpi for RM. Total RNA was purified using the RiboPure™ kit (Ambion, Austin, USA) following the manufacturer’s recommendations, then treated twice with DNAse I (Ambion), precipitated with ethanol and resuspended in RNAse-free water. Nine μg of total RNA was subjected to two successive rRNA removal steps using the Microbe Express™ kit (Ambion) to finally obtain 1 μg of enriched messenger RNA. The quantity and quality of total and enriched RNA were estimated using a NanoDrop ND-1000 and Bioanalyser Agilent 2100 (RNA nanochip), respectively (Additional file 1: Figure S13). All total and enriched messenger RNA samples were characterized by RIN values ranging from 9 to 10 indicating high quality.
Eighteen RNA samples (3 bacteria × 2 conditions × 3 biological repetitions) were sent for synthesis of corresponding cDNA libraries and sequencing to GATC Biotech (Konstanz, Germany). cDNA libraries were prepared according to the manufacturer’s instructions (Illumina TruSeq™ RNA Preparation) and labelled to sequence 2 to 3 libraries per lane (at least 50 million reads per library, read length of 36 bp) on a Genome Analyzer II (Illumina). Raw data sequences were submitted to ArrayExpress (www.ebi.ac.uk/arrayexpress) under accession: E-MTAB-4144.

Total RNA was isolated from urethral swab and cervicovaginal lavage specimens using TRIzol (Invitrogen) as described previously (38 (link)). Briefly, specimens were washed twice with 70% ethanol and treated with Turbo DNase (Ambion), the RiboMinus kit (Invitrogen) (to deplete eukaryotic rRNA), and the Microbe Express kit (Ambion) (to deplete bacterial rRNA) using diethylpyrocarbonate (DEPC)-treated EDTA-free water. Gonococcal isolates that corresponded to each specimen were grown overnight on chocolate agar plates at 37°C in 5% CO₂ prior to inoculation in chemically defined medium (CDM) containing ferric nitrate (100 µM). Liquid cultures were inoculated at an optical density at 600 nm (OD₆₀₀) of 0.1, harvested after 3 h, and pelleted, and RNA was extracted as described above.

Cultures for each strain and treatment group were prepared using independent biological quadruplicates. Wild-type (OG1) and ΔcroR mutant (SB23) were grown to exponential-phase in MHB at 37°C and aliquots were treated with bacitracin (128 μg/mL) to activate the CroS/R TCS. After 15 min of treatment (intended to capture the initial transcriptional response to cell wall stress rather than secondary effects of growth inhibition), cultures were harvested by mixing with equal volumes of acetone/ethanol (1:1) as described above. RNA was prepared as described above and subjected to rRNA depletion using the MicrobeExpress kit according to the manufacturer’s instructions (ThermoFisher). Library preparation and sequencing were performed at the University of Minnesota Genomics Center.