Lightcycler instrument

The total RNA from different tissues and whole seedlings under different treatments were extracted using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China). The RNA concentration and purity were determined using the NaNo drop 1,000 spectrophotometer and agarose gel electrophoresis (Xie et al., 2018 (link)). The FastQuant first strand cDNA synthesis kit (TIANGEN, Beijing, China) was used for the synthesis of cDNA following the manufacturer’s protocol. The SuperReal PreMix Plus kit (TIANGEN, Beijing, China) and a Roche LightCycler instrument were used for qRT-PCR. The reaction system of qRT-PCR was as follows: 10 μL 2×SuperReal PreMix Plus, 0.6 μL 10 μM forward primers, 0.6 μL 10 μM reverse primers, 2 μL cDNA and 6.8 μL RNase-free ddH₂O. The qRT-PCR procedure was as follow: 95 °C for 15 min and 40 cycles of 95 °C for 10 s and 60 °C for 20 s. CsActin was used as an internal reference gene (Zhou et al., 2017 (link)). The primers of the cucumber TPS genes and CsActin for qRT-PCR were designed and synthesized using Sangon Biotech online software (Table 1). Three technical replicates were performed for each reaction.

Total RNA was extracted from GC tissues and their ANTs using Trizol reagent (Invitrogen, Karlsruhe, Germany), and reverse transcription reactions were performed using the GoScript Reverse Transcription (RT) System (Promega, Madison, USA) according to the manufacturer's instructions. Real-time PCR was performed using a standard SYBR Green PCR kit (Roche, USA) and a Roche Light^® Cycler Instrument (Roche, USA) according to the respective manufacturer's instructions. β-actin or GAPDH was used to normalize lncRNAs. Each sample was analyzed in triplicate. Primers for SLC7A11-AS1, SLC7A11, ASK1, cyclin D1, Gclm, c-Jun, p38, β-actin and GAPDH were synthesized by Sangon Biotech (Shanghai, China), and their sequences are listed in Supplementary Table 1. The thermal cycling consisted of a denaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 63°C or 60°C for 30 s, and extension at 72°C for 60 s. At the same time, β-actin or GAPDH was amplified for normalization of the relative levels of lncRNA. Amplified DNA products were subjected to separation by 1.5% agarose gel electrophoresis and staining with ethidium bromide for visualization. The 2^-ΔΔCt method was used to quantify the relative levels of gene expression.

Total RNA was isolated from hippocampal CA2, CA3, and hypothalamus tissue lysates using a Tri Reagent kit (Molecular Research Center) and treated with RNase-free DNase (RQ1; Promega) to remove potential contamination by genomic DNA. Total RNA (2 μg) from samples was reverse transcribed using a SuperScript cDNA synthesis kit (Invitrogen). qPCR was performed on the Roche LightCycler instrument (Roche Diagnostics) using the FastStart DNA Master SYBR Green I kit (Roche Applied Science) according to the manufacturer’s instructions. The primers used in this experiment for Oxtr were as follows: forward (5′-TTCTTCGTGCAGATGTGGAG-3′) and reverse (5′-CCTTCAGGTACCGAGCAGAG-3′). The PCR reactions were run for 40 cycles. Each amplification cycle included denaturation at 95 °C for 20 s, annealing at 58 °C for 20 s, and extension at 72 °C for 40 s. All reactions were repeated in duplicate and data were analyzed by lightcycler relative quantification software. The expression levels of the target gene were normalized to β-actin rRNA.