Bullseye evagreen master mix

B. burgdorferi cultures were pelleted at 4°C, 3,200 x g for 20 minutes. Cell pellets were washed once with HN buffer (10 mM HEPES, 10 mM NaCl, pH 8.0). RNA isolation was performed using RNAzol (Sigma-Aldrich, St. Louis, MO, United States) according to kit instructions. The RNA was quantified by spectrophotometry using a TAKE3 plate in a Cytation 5 multi-mode plate reader (Biotek, Winooski, VT, USA), and RNA quality was assessed by analysis of ribosomal RNA bands visualized following gel electrophoresis by SYBR-safe dye incorporation. cDNA was generated from 500 ng of total RNA from each sample with the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, Foster City, CA, United States) following kit instructions. RT-qPCR was performed on the cDNA in the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, United States) using Bullseye EvaGreen Master Mix (MIDSCI, St. Louis, MO, United States) reagents and oligonucleotide primers targeting the gene of interest (S1 Table). The RT-qPCR data were analyzed using the ΔCq method to indicate copies per 16S rRNA transcript levels.

cDNA synthesis was performed with approximately 1 µg of RNA with the RNA high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). The quantitative PCR (qPCR) amplification was performed in Bullseye EvaGreen master mix (MIDSCI, Valley Park, MO) using oligonucleotide primers specific to the gene of interest (Table S5) and detected using the CFX Connect real-time PCR detection system (Bio-Rad, Hercules, CA). All quantification cycle (C_q) values were calculated by the CFX regression method. The C_q values of raw RNA inputs into the cDNA reaction (minus the reverse transcriptase [RT] control) ensured that samples were DNA free. The 16S rRNA transcript levels were utilized as the reference. Typically, rRNA levels are significantly reduced during the stringent response, and DksA in E. coli has specifically been implicated in controlling the expression of rrnB1; however, the C_q values of 16S rRNA were less responsive to various conditions and strains than other commonly used B. burgdorferi reference genes, such as flaB and rpoD (Fig. S4). The RT-qPCR data were analyzed in Excel (Microsoft, Redmond, WA) using the ΔC_q method to represent transcript levels relative to 16S rRNA. Graphing and statistical comparisons were performed with Prism (GraphPad, La Jolla, CA).