Primer 5 program

To verify the reliability of RNA-seq, 16 DEGs involved in cold response were selected for qRT-PCR. The primer pairs (Table S1) were designed with the Primer 5 program (version 5.0, Premier Biosoft International, Palo Alto, CA, USA), and PP2A-1 was selected as the internal standard control [67 (link)]. Total RNA was used for first cDNA strand synthesis with the PrimeScriptTM reagent kit (Takara, Dalian, China). The mRNA expression was quantified using the SYBR Premix Ex Taq II kit (TaKaRa, Dalian, China). A quantitative real-time polymerase chain reaction (qRT-PCR) was conducted on a StepOnePlus™ real-time PCR system (Applied Biosystems, Foster City, CA, USA). Three biological and technical replicates for each gene were employed. Relative gene expression levels were calculated using the 2^−ΔΔCT method [68 (link)].

The expression patterns of eight randomly selected genes encoding the identified metabolites were analysed with primer pairs (Table 1) designed with the Primer 5 program (Premier Biosoft International, California, USA). cDNA was synthesised using 2 μg of total RNA (DNA-free), RevertAid reverse transcriptase (Thermo Scientific, Waltham, MA, USA) and Random Hexamer Primers (5′-NNNNNN-3′; N = G, A, T or C) (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s protocol. The qPCR assay was conducted on a Rotor-Gene Q System (Qiagen, Hilden Germany) based on a method described previously^{51 (link),52 (link)} using SsoAdvanced Universal SYBR Green Supermix (2×) (Bio-Rad, Hercules, California, USA). The mixtures were subjected to an initial step at 95 °C for 1 min, followed by 40 cycles of denaturation at 95 °C for 20 s, annealing at 60.5 °C for 20 s, and elongation at 72 °C for 15 s. Melting curve analysis was performed by heating the amplicon from 72 °C to 95 °C. Relative gene expression levels were calculated according to the 2-ΔΔCT method, with ef1-α and rpl2 for T. rubrum, and adp-rf and mbp-1 for M. canis (Table 1) as the reference genes according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines^{56 (link)}.