Rneasy bacteria mini kit

Bacterial RNA extractions were carried out from in vitro-cultured cells after 18 h and from cells grown in planta at different time intervals.
For RNA isolation from in vitro cultured bacteria, Pst DC3000 was grown on KB agar plates for 48 h at 28°C. Hx was added to 1×10⁶ bacterial cells suspensions to final concentrations of 1.5 mM, 5 mM and 10 mM. Cells were incubated by shaking for 18 h before RNA extraction. Bacterial suspensions were stabilized with RNA protect bacterial reagent (Qiagen) before centrifugation. Cells pellets were immediately frozen in liquid N₂ and kept at −80°C prior to RNA extraction. We used the Qiagen RNeasy Bacteria Mini Kit for isolation of RNA samples according to manufacturer's instructions.
To extract bacterial RNA from infected plants we used a protocol for extracting RNA from P.syringae recovered from infected leaves as described by Yu et al. [55] (link). RNA quantification was performed with a UV-VIS UNICAM Model Heλiosβ spectrophotometer. Reverse transcription was carried out from 1 µg of total RNA for gene expression analysis, by using OMNISCRIPT (Qiagen) and random hexamer primers (Promega).

For isolation of RNA, 500 μl of a S. pneumoniae culture at an OD₆₀₀ of 0.3, grown in C+Y media at 37°C with 5% CO₂, was mixed with 1 ml of RNA Protect (Qiagen). RNA was extracted and purified using an RNeasy Bacteria Mini Kit (Qiagen) after enzymatic lysis using lysozyme and mutanolysin, as described previously (Eijkelkamp et al., 2014a (link), 2015 (link), 2016 (link); Plumptre et al., 2014a (link),b (link); Begg et al., 2015 (link)). The total RNA samples were treated with DNase I (Roche) and qRT-PCR was carried out using a SuperScript III One-Step RT-PCR kit (Thermo Fisher Scientific) on a LC480 Real-Time Cycler (Roche). Transcription levels of genes analyzed were normalized to those obtained for 16S rRNA. Primer sequences are available in Table S1. Results are representative of at least four independent samples and the statistical difference was examined by an unpaired Student t-test (Graphpad Prism 6.0c).