Bacterial qPCR Analysis Under Iron Stress

For qPCR studies, bacteria were streaked from glycerol stocks onto LB plates; then, single colonies were cultured in 4AA broth. Overnight cultures were diluted 100-fold into fresh 4AA broth with or without deferoxamine (100 µM) and harvested at an OD₆₀₀ of 0.5. Cells were treated with RNAprotect (Qiagen 1018380, Hilden, Germany); then, RNA was purified via repeated phenol-chloroform extraction. The resulting nucleic acid precipitate was resuspended in nuclease-free H₂O, digested with DNAse I (Ambion AM2222, Austin, TX, USA), and purified using Qiagen’s RNeasy Plus kit (Qiagen 74134). cDNA was prepared using 250 ng–1000 ng RNA via iScript kit (BioRad 1708891, Hercules, CA, USA) before being diluted at 1:40 for qPCR analysis. qPCR reactions were carried out using iTaq Universal SYBR Green Supermix (BioRad 1725121) on a CFX Connect according to the manufacturer’s protocol. Fold-change was determined via the ΔΔCt method, whereby each gene of interest was calibrated to the 16S ribosomal housekeeping gene. Primers used for qPCR analysis can be found in Supplemental Table S2.