On column dnase 1 digestion kit

Total RNA from BL21(DE3) and DH5α E. coli (≈5x10 8 cells) was stabilized using the RNA-protect Bacteria Reagent (Qiagen, Izasa Scientific, Barcelona, Spain). RNA was isolated after lysozyme-proteinase K digestion using RNeasy Mini Kit (Qiagen) and On-Column DNase I digestion kit (Qiagen). RNA concentrations were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Madrid, Spain) and RNA quality was evaluated on a denaturing agarose gel. Reverse transcription (RT) was carried out using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain). RT reactions without retrotranscriptase were used as the controls to confirm the lack of contaminating DNA in the RNA samples. The cDNA obtained was used for quantitative PCR (qPCR) in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) using SYBR Green I (Applied Biosystems), as previously described [27] .
The oligonucleotide sequences of the primers and the thermal cycling conditions used for measuring the abundance of TolC mRNA and 16S rRNA, used as the normalizer, are shown in the Supplementary Table 1.