RNA Extraction and Quantification in M. genitalium

RNA was extracted from mid-log phase cultures of M. genitalium using the RNAqueous Kit (Thermo Fisher Scientific) and then treated with Turbo DNase (Thermo Fisher Scientific) following the manufacturer’s instructions. When necessary, cultures were treated with the iron chelator 2,2-bypiridyl (1 mg ml⁻¹) (Sigma-Aldrich) for 1 h before the cell lysis to create a transition metal limited environment. Reverse transcription was performed with iScript Reverse Transcriptase (Bio-Rad) and random primers as previously described [21 (link)]. Primers used for qPCR are listed in Supplemental Table S3 and they were designed using Primer3 software. qPCR was performed with iTaq polymerase (Bio-Rad) and SYBR green in CFX96 or CFX384 PCR instruments (Bio-Rad). Relative gene expression was calculated using the Pfaffl method [27 (link)]. Differential gene expression was judged based on the common arbitrary 2-fold cutoff. Data presented in the manuscript correspond to the analysis of RNAs isolated from three independent biological repeats.