Fqd 96a

Liver samples were used to obtain total RNA firstly. After checking RNA quality, they were consigned to Shanghai Personal Biotechnology Co., Ltd. for constructing RNA-seq libraries and sequencing using Illumina NovaSeq. In addition, extracted RNA was reverse transcribed into cDNA for verifying genes expression by RT-PCR method using SYBR Green Pro Taq™ II kit (Agbio, Hunan, China) on the FQD-96A (Bioer, Hangzhou, China). Primers were shown in Table S1. Detailed data processes such as differential expression genes identification, GO and KEGG pathways analysis and calculation of gene expression all referred to our previous report [25 (link), 26 (link)].

Total RNA was extracted using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific, Amercia) and quantified by NANODROP ONE (Thermo Fisher Scientific, Amercia). One µg of total RNA was then reverse transcribed to cDNA using the TAKARA RR047A PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time). Gene expression levels were measured using a qPCR machine (BIOER, FQD-96A, HangZhou) with MonAmp™ ChemoHS qPCR Mix (Monad, ShangHai). The relative expression level of NADK was calculated using the 2^−ΔΔCt method with GAPDH as the internal control. The primer sequences are as follows: NADK forward: 5′-ACCTGAAGCAAGGAACACAGC-3′; NADK reverse: 5′-AGCGGGTAGCATGAGGTAGT- 3′. GAPDH forward: 5′-GCTGAGAACGGGAAGCTTGT-3′; GAPDH reverse: 5′ -GCCAGGG GTGCTAAGCAGTT-3′.

Total RNA was extracted from leaf samples according to the instructions of Biofit kit (Tsingke, China). The strand cDNA was synthesized using Goldenstar RT6 cDNA synthesis kit (Tsingke, China). The expression levels of representative unigenes and selected key genes in the flavonoid biosynthesis pathway were analyzed by qRT-PCR using FQD-96A (Bioer Technology, China). The 20 μl reaction mixture contained 10 μl SYBR^® Green Real-time PCR Master Mix (CWBIO), 1 μl cDNA template, 0.8 μM of each forward and reverse primer, and 7.4 μl ddH₂O. The following cycling parameters were applied for amplification: 95°C for 1 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, then followed by 95°C for 5 s, 60°C for 1 min, 0.11°C / s to 95°C, 50°C for 30 s for plate reading. The primers list is shown in Supplementary Table S1. The actin gene was selected as an internal reference (Xu et al., 2019 (link)). Three replicates were performed for each sample. Quantitative data were analyzed using the 2^−ΔΔCT method.