Sybr mix

Total RNA and protein were extracted using TRIzol reagent (Sigma) according to the manufacturer′s instruction. For cDNA synthesis, 1 μg total RNA was reverse-transcribed into cDNA using the FastQuant RT Kit (TIANGEN). Then quantitative PCR was performed using 2 × SYBR mix (Vazyme), and the reaction was run on the Bio-Rad CFX manager machine. The sequence information of all primers is listed in Supplementary Data 2.

Quantitative real-time PCR (qRT-PCR) analysis was performed with a CFX96TM Real-time System (BIO-RAD, Hercules, USA) with 10 μL of SYBR Mix (VAZYME, Nanjing, China), 6 μL of ddH₂O, 1 μL of each primer, and 2 μL of cDNA template to a final volume of 20 μL. The implemented reaction procedure was 95 °C for 60 s, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The relative gene expression levels were calculated using the 2^−ΔΔCT method [63 (link)] by normalization to GmACTIN (Glyma.19G147900⁾ [64 (link)] or AtACTIN7 (AT5G09810) in soybean and Arabidopsis, respectively [65 (link)]. All the primers used in this experiment are listed in Supplementary Table S1.

Trizol was used to extract total RNA from the cells. cDNA was synthesized through reverse transcription (Vazyme, Shanghai, China) using the BIO-RAD instrument and with SYBR Mix (Vazyme, Shanghai, China) to perform qRT-PCR.The information on the sequence is provided in Supplementary MaterialTable S2.

A total of 0.5 g of plant materials was ground in liquid nitrogen, mixed with 1 mL of TRIzol (Invitrogen, Shanghai, China), and then extracted with 200 μL of chloroform. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and mixed with an equal volume of isopropanol. The precipitated RNA precipitation was obtained after centrifugation at 12,000 rpm for 10 min. The RNA was extracted after washing with 75% ethanol. RNA was reverse-transcribed into cDNA (Vazyme, Nanjing, China, R333), and qRT-PCR was performed according to the SYBR Mix instructions (Vazyme, Nanjing, China, Q411). qRT-PCR was performed using LightCycler96 (Roche, Rotkreuz, Switzerland). The potato EF1α gene and the N. benthamiana actin gene were used as the internal reference genes. The internal reference gene and detection primers used in qRT-PCR are shown in Supplementary Table 1. Data were collected from three biological replicates, and the bar value represents the standard deviation. Statistical analysis of the two sets of data was based on the Student’s t-test, ^nsp > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Total RNA was extracted using a commercial kit (Aidlab, China), and the purity and amount of RNA were assessed using NanoDrop 2000 (Thermo Fisher Scientific, USA). 1ng RNA was used for reverse transcription with HiScript II mix (Vazyme, China), and qPCR was performed with SYBR mix (Vazyme, China) according to the manufacturer’s protocol. GAPDH was selected as the reference gene. The primer sequences of GAPDH were 5′-CAT​CAT​CCC​TGC​CTC​TAC​TGG-3′ (forward) and 5′-GTG​GGT​GTC​GCT​GTT​GAA​GTC-3′ (reverse), and the primer sequences of SNX14 were 5′-TCC​CCG​AAA​CCT​TGC​TGC-3′ (forward) and 5′-GGC​TCG​TGT​CCA​ACT​GCT-3′ (reverse). The 2^−ΔΔCt method was used to calculate relative expression.

Trizol reagent (Invitrogen) was utilized to obtain total RNA from melanoma tissues and cells. Then a HiScript III First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) or a miRNA First Strand Synthesis Kit (Vazyme) was used to conduct the reverse transcription reaction. Next, the amplification reaction was performed using SYBR Mix (Vazyme) and a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative expression was analyzed using the 2^−ΔΔCt method and standardized by GAPDH or U6. The primer sequences were as follows: circ_0084043, F: 5′-TTCTAGACAGCCGGGGAGTG-3′ and R: 5′-CCAAAACCTTTCTTTCTTGATGGGA-3′; miR-31, F: 5′-GCCGCAGGCAAGATGCTGGC-3′ and R: 5′-CAGTGCAGGGTCCGAGGT-3′; KLF3, F: 5′-TGTCTCAGTGTCATACCCATCT-3′ and R: 5′-CCTTCTGGGGTCTGAAAGAACTT-3′; GAPDH, F: 5′-ACCACAGTCCATGCCATCAC-3′ and R: 5′-TCCACCACCCTGTTGCTGTA-3′; U6, F: 5′-GCGCGTCGTGAAGCGTTC-3′ and R: 5′-GTGCAGGGTCCGAGGT-3′.