Hybond n hybridization membranes

A northern blot assay was carried out as described previously [32 (link)]. Total RNA was prepared from S. aureus culture samples using miRNeasy Kits (Qiagen). Electrophoresis of total RNA (5-10 μg) was performed onto a polyacrylamide gel (PAGE) containing 7% urea (1.5 h, 120 V, 4°C). After migration, RNAs were vacuum transferred on Hybond®-N+ hybridization membranes (Cytiva). Hybridization with specific biotin-labeled probes complementary to RSaX28 sequences, followed by luminescent detection, was carried out as guidance of the North2South™ Chemiluminescent Hybridization and Detection Kit (Thermo Scientific).

In vitro transcription and EMSA assays were carried out as described previously [32 (link)]. The PCR fragment of RSaX28_WT was amplified using primers T7-RSaX28-F and T7-RSaX28-R, and RSaX28_mut was amplified using T7-RSaX28-F, T7-RSaX28-mut2, T7-RSaX28-mut1 and T7-RSaX28-R, so as to the PCR fragments of RNAIII_WT and RNAIII _mut. Transcription of T7 RNA was performed using RiboMAX Large Scale RNA Production Systems (Promega) with or without Biotin-16-UTP (Roche) as described in the Guidance. The RNAs were then purified by using miRNeasy RNA extraction kits (Qiagen) followed by dissolving in TMN buffer (20 mM Tris-acetate at pH 7.5, 10 mM magnesium acetate, 150 mM sodium acetate, 1 mM DTT). Biotin-labelled purified RSaX28_WT or RSaX28-mut (50 nM/sample) and cold RNAIII_WT or RNAIII_mut (0 nM, 50 nM, 100 nM, 200 nM/sample) were mixed in TMN buffer in a total volume of 10 uL. Binding was performed at 37°C in TMN buffer for 10 min. After incubation, RNA loading buffer was added and electrophoresis of the samples were performed onto a polyacrylamide gel (PAGE) under nondenaturing conditions (1 h, 100 V, 4°C). After migration, RNAs were transferred onto Hybond®-N+ hybridization membranes (Cytiva), followed by luminescent detection were carried out using North2South™ Chemiluminescent Hybridization and Detection Kit (Thermo Scientific).