cDNA synthesis and RT-qPCR.

cDNA was synthesized from 1 μg of total bacterial RNA using Superscript III reverse transcriptase (Invitrogen, USA), RNase-Out (Invitrogen, USA), and random nonamer primers. Reverse transcription was performed from 1 μg of total RNA. All reagents were purchased from Invitrogen (USA). Sufficient quality of cDNA was ensured by control PCRs (amplification of 16S for bacterial cDNA).

RT-qPCR was routinely performed on 0.5 μL of cDNA in a CFX96 real-time PCR cycling machine (Bio-Rad, USA) using H. pylori gene-specific primers (synthesized at Metabion) (Table 5) and 2× SYBR Green master mix (Qiagen, Hilden, Germany). Transcript quantification was always performed in triplicate, with gene-specific standards for absolute amount quantification, and performed in parallel for each transcript using the following protocol: 95°C for 10 min; 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 40 cycles; and a melting curve of 60°C to 95°C with an increment of 0.5°C for 5 s. Results were equalized to 1 μL of cDNA and normalized to transcript amounts of the H. pylori 16S gene using a correction factor respective to the mock reference but maintaining the absolute values (pg/μL) for all final transcript amounts. MiQE standards for the RT-qPCR method were applied as detailed in reference 37. Statistics for Fig. 1C to ​toFF are provided in Table S1 in the supplemental material. Each figure shows different biological experiments, which explains the slight variation of absolute transcript quantities. Different experiments can have slightly different starting quantities/transcript amounts of certain transcripts due to biological variation.

List of gene-specific primers used for RT-qPCR