Rtase

To confirm the transcriptome data, the quantitative real-time PCR (qPCR) was performed for the selected genes. Totally, 16 pairs of primers were designed (Additional file 8: Table S5) and the 16 sRNA gene was used as the control. The RNAs were isolated from the same cell samples as the transcriptome analysis. The reverse transcription (RT) was performed in 20 μL mixture including 1 μg RNA sample and 5 U RTase (Takara, Dalian, China). The resulting cDNA was diluted by × 10 folds and used as a template for qPCR. qPCR was carried out following the instruction of 2 × Real Time PCR EasyTM-SYBR Green I Kit. The calculated cycle threshold (CT) was normalized to the CT of 16 sRNA amplified from the corresponding sample. Changes in mRNA levels were calculated using the 2^-ΔΔCT method [61 (link)].

Total RNAs were reverse transcribed to cDNAs using an RTase (Takara Bio, Kusatsu, Japan), following the manufacturer’s instructions, and subjected to qPCR as described in Zhang, et al.^{27 (link)}. Primers used for qRT-PCR was listed in Table S4.