Quantitative PCR validation of RNA-seq data

RNA-seq results were validated using qPCR amplification of three genes: 1) leuA which exhibited >4-fold higher expression at 4°C at all five growth phases, 2) cspB which exhibited >4-fold lower expression at 4°C at all growth phases, and 3) recJ which was chosen as a reference gene because it showed very little variation in expression across all growth phases at either temperature (S2 Table). Up to 1 μg of RNA from each of our 30 samples was reverse transcribed using the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA) per the manufacturer’s protocol. Primers were designed using Primer3 Plus (Table 1) and our draft whole genome sequence for Lm1 (GenBank Accession number GCA_001709805.1). qPCR was conducted in a CFX96 Touch^™ Real-Time PCR Detection System (BioRad) using SsoAdvanced ^™ Universal SYBR Green ^® Supermix (BioRad). The relative expression levels of leuA and cspB were calculated using the 2^-ΔΔCT method [49], with recJ as the reference gene.