Validation of Differential Expression by RT-qPCR

The validity of differential expression was verified by using RT-qPCR for direct comparison with RNA Seq. The qPCR reactions (10 uL) were performed in triplicate using iTaq Universal One-Step RT-qPCR kit (BioRad, Hercules, CA, USA) with 0.5 μM of each primer (Supplementary Table 2), and 1 ng of total RNA as template. First cDNA was synthesized by reverse transcription at 50 °C for 10 min followed by reverse transcriptase inactivation at 95 °C for 1 min. The reaction was directly followed by PCR amplification as follows: 40 cycles of denaturation: 30 s at 95 °C; annealing: 30 s at 55 °C; and extension: 30 s at 72 °C. The final PCR step was 30 s at 96 °C followed by 5 s at 60 °C and the PCR reaction was stopped by a constant temperature of 4 ℃. The relative expression of the target genes was calculated using gyrA and rpoA as reference genes and using the efficiency-corrected REST model [37 (link)], CFX Maestro software version 2.2 (BioRad) and qbase + version 3.3 [36 (link)]. The gyrA and rpoA genes were chosen using reference gene selection tool CFX Maestro software version 2.2 (BioRad). For each comparison, four biological replicates and three technical replicates were used for all calculations.