Bacteria rna protect

Bacteria were grown overnight on GCB supplemented with or without IPTG (Sigma-Aldrich) for 18–20 hours and swabbed into Bacteria RNA Protect (Qiagen), pelleted, and frozen at −80°C. RNA was isolated via the RNeasy Mini Kit (Qiagen) and treated with DNase I (Promega) to remove contaminating gDNA. RNA was incubated with Random Hexamers (Promega) and subsequently reverse transcribed with the SuperScript III Reverse Transcriptase (Thermo Fisher). A no-RT control was made for each sample to ensure that there was minimal to no gDNA contamination. The qPCR primers used for this study were designed with the IDT Primer Design tool and are listed in Table S1. qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) in a Bio-Rad iQ5 Real-Time Detection system. The Gc gene, rmp, was used as an internal control (99 (link)). Gene expression change was calculated using the 2^−ΔΔCT cycle threshold method (100 (link)). qRT-PCR experiments were conducted following the Minimal Information for Publications on Quantitative Real-Time PCR Experiments guidelines (101 (link)).