Itaq polymerase

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. Reverse transcription was performed with iScript Reverse Transcriptase (Bio-Rad) and random primers as previously described (Torres-Puig et al., 2015 (link)). Primers used for qPCR are listed in Supplementary Table S2 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 (Pfaffl, 2001 (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.

Hybridization probes developed as previously described [52 (link)], although with some modifications. As described above, total RNA was isolated from frozen MPOA. Total RNA level was adjusted to 2 µg/µL for each sample. Then, cDNA was synthesized and amplified with iTaq polymerase (BioRad, Hercules, CA, USA). The primers were the same as above for RT-PCR. The PCR products were purified from gel and applied as templates in PCR reactions, using the same primers specific for Ndufs5, Nwd1, and Rbm3 except the reverse primers were supplemented with T7 RNA polymerase recognition site. PCR products were purified from gel again and used as templates in subsequent PCR reactions with the same forward and T7-containing reverse primers. Products of these reactions were purified and used to synthesize labelled RNA probes for in situ hybridization.

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.