Realmastermix

Cells were seeded in six-well plates and cultured. Total RNAs were extracted from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Gene expression was verified by using AZpolarisTM Cdna Synthesis Kit (Azanno Biotech, Gothenburg, Sweden) for reverse-transcription and RealMaster Mix (Tiangen Biotech, Beijing, China) for reverse-transcription-PCR, according to the manufacturer’s instructions. The primers used were as follows: BNIP3-FWD (5′-CAGGGCTCCTGGGTAGAACT-3′), BNIP3-REV (5′-CTACTCCGTCCAGACTCATGC-3′); GAPDH-FWD (5′-TTGGTATCGTGGAAGGACTCA-3′) and GAPDH-REV (5′-TGTCATCATATTTGGCAGGTT-3′).

Randomly chosen DEGs were used for the validation of RNA-seq results by qRT-PCR. Primer 6.0 software was used to design the gene-specific primers and synthesized by Sangon Biotech Company (Shanghai, China). The expression patterns of the chosen lncRNA and mRNAs were studied through qRT-PCR using RealmasterMix (SYBR Green, TIANGEN, China) following the manufacturer’s instructions. Gapdh mRNA was used as an internal control. The method 2^−ΔΔCt method was used for the quantitative results to interpret the fold changes, followed by the statistical analysis. All the designed primer sequences are given in Table S1.

Total mRNA was isolated using Trizol reagent according to the manufacturer's instructions (Invitrogen). Complementary DNA (cDNA) was synthesized by reverse transcription using Invitrogen Superscript reagents according to the manufacturer's instructions. Primers used in qRT-PCR are as follows: c-Jun F, 5′-TCG ACA TGG AGT CCC AGG A-3′; c-Jun R, 5′-GGC GAT TCT CTC CAG CTT CC-3′; Cyclin D1 F, 5′-GCG CGT ACC CCG ATG CCA AC-3′; Cyclin D1 R, 5′-CTC GCA GAC CTC CAG CAT CCA-3′; β-actin F, 5′-GGA TGC AGA AGG AGA TCA CTG-3′; β-actin R, 5′-CGA TCC ACA CGG AGT ACT TG-3′. qRT-PCR was performed with RealMasterMix (SYBR Green; Tiangen FP202) using a BioRad iQ5 (BioRad). Housekeeping gene β-actin was used as an internal control for normalization.

qRT-PCR was used to analyze AnGolS1 transcript accumulation in different tissues of A. nanus seedlings in a greenhouse under different stresses grown. Total RNA was isolated from leaves, stems, and roots by using the TaKaRa MiniBEST Universal RNA Extraction Kit (Takara, Japan), and first-strand cDNA was synthesized as mentioned above. This cDNA was used as a template in qRT-PCR with the primers AnGolS1 RT-F and AnGolS1 RT-R, which are specific to the AnGolS1 coding sequence. Actin-F and Actin-R primers were used to amplify the housekeeping gene Actin as an internal control. The qRT-PCR reactions were performed in triplicate for each sample using Real Master Mix (SYBR Green) (Tiangen, China) on a real-time PCR system (ABI 7500, USA) under the following conditions: 95 °C for 60 s, 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 68 °C for 60 s, 1 cycle of 95 °C for 15 s and 60 °C for 1 min, and 95 °C for 15 s. The specificity of each primer pair was validated by a dissociation curve. The experiment was repeated at least three times, and gene expression levels were calculated based on the method described by Pfaffl57 (link). Significant differences in the data were analyzed according to Duncan’s multiple range test.

Total RNA was reverse transcribed using random primer in a 20 μL reaction mixture. Relative mRNA levels of candidate ISGs were measured by SYBR Green real-time PCR (RealmasterMix; SYBR Green; Tiangen) in Rotor-gene 6000 (Corbett Life Science) using the following program: (i) 95°C 10 min, 1 cycle; (ii) 95°C 15 s; 60°C 30 s; 72°C 30 s, 40 cycles. All data are shown as mean value for at least two independent measurements. GAPDH mRNA levels served as internal control. Primers (Table 1) used for the PCR assays are designed using the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) or acquired from Primer Bank (https://pga.mgh.harvard.edu/primerbank/). Amplification efficiency was assessed for all primer sets, and primers with efficiencies 90%–110% were used.