Rt reagent

RNA was isolated from tumor and paratumor tissues using an RNA extraction kit (Promega, Beijing, China). Single-stranded cDNA was generated from 1ug total RNA in a 20 μl reaction volume with 4 μl RT reagent (Takara, Japan). The quantitative real-time PCR reaction was performed with the SYBR green detection (Penzberg, German). GAPDH was used as an endogenous control. The relative expression levels were measured by qRT-PCR using LightCycle 480 II (Roche, Switzerland). Each of the experiments was performed in triplicate. The primer pairs for each target gene were listed in Additional file 1.

The total RNA of single cell suspension were extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and the complementary DNA (cDNA) was synthesized using reverse transcription (RT) reagent (Takara, Dalian, Liaoning, China), following the kit instructions. The RNA was then amplified using PCR as follows: 95 °C for 8 min, then 30 cycles of 95 °C for 20 s, 65 °C for 35 s, and 70 °C for 30 s, after which the RNA was denatured at 90 °C for 20 s, 55 °C for 1 min, then stored at 4 °C. A 1.5% agarose gel electrophoresis was used to detect the integrity of 28S and 18S bands, as well as their ratio; the concentration was determined using a ThermoScientific NanoDrop 2000c (ThermoFisher Scientific Inc., Waltham, MA, USA). Only samples with 1.8 < OD 260/280 nm < 2.0, OD 260/230 nm ≥ 2.0, RNA Integrity Number: RIN ≥ 6.5, and 28S/18S ≥ 1.0 were used for subsequent analyses, and the PCR primers are listed in Supplementary Table S1.

Total RNA from cells was extracted using TRIzol reagent (Life Technologies, CA, United States) following the manufacturer’s protocol. For quantitative real-time RT-PCR, cDNA synthesis was performed using 500 ng RNA per sample using RT reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. qRT-PCR amplification was performed on a StepOnePlus real-time PCR system (Applied Biosystems, CA, United States), and data were analyzed using the 2^−ΔΔCT method, with GAPDH RNA as an endogenous control. The primer sequences were as follows: KNL1, forward 5′-ACC​TCT​CTG​GAC​TTC​AGC​ACT​TAC​C-3′ and reverse 5′-TCT​GTA​TCA​AGA​TGT​GGA​CCT​GGA​G-3′; GAPDH, forward 5′-ATG​GTG​AAG​GTC​GGT​GTG​AA-3′ and reverse 5′-GAG​TGG​AGT​CAT​ACT​GGA​AC-3′.

G. hirsutum (CCRI 12–4) and G. darwinii 5-7 species were used in this research to investigate gene expression under 200 mmol salt treatment at three leaf stages while grown in a hydroponic system. Leaf samples were taken after 0 h, 1 h, 3 h, 6 h, and 24 h of salt stress treatment and quickly transferred into liquid nitrogen and stored at –80°C for RNA extraction. Under controlled conditions, seedlings were treated with only fresh water. The RNAprep Pure Plant Kit (Tianjin, China) was used for the extraction of RNA from leaf samples. The RNAprep Pure Plant Kit was used for the extraction of RNA from leaf samples. Single-stranded complementary DNA (cDNA) was synthesized using the RT reagent Takara kit, China. Protein kinase gene primers were designed from the online NCBI database. Supplementary Table S6 contains primer information. RT-qPCR was carried out in accordance with the method described by Wang et al. (2020) (link).

Total RNA was isolated from the jejunal mucosa by using TRIZOL reagent (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) according to the manufacturer’s instructions. The RNA quality was determined by DU 640 UV spectrophotometer detection (Beckman Coulter Inc., Fullerton, CA), and the OD₂₆₀:OD₂₈₀ ratio ranged from 1.8 and 2.0 in all samples. The RNA integrity was analyzed by 1% agarose gel electrophoresis. The RNA samples were reversely transcribed into complementary DNA by using RT Reagents (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) according to the manufacturer’s instruction. Following reverse transcription, expression levels of ZO-1, occludin, Bcl-2, Bax and β-actin in the jejunal mucosa were analyzed by real-time quantitative PCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA) as previously described [32 (link)]. The primers were purchased by TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China), which was listed in Table 2. Relative gene expression to the reference gene (β-actin) was determined in order to correct for the variance in amounts of RNA input in the reaction. In addition, the relative gene expressions compared to the reference gene were calculated with the previous method [33 (link)].

RT-qPCR was used to verify the expression levels of the differentially expressed genes and miRNAs. Approximately 0.1 μg of RNA per sample was reverse transcribed into cDNA using RT reagents (Takara, Dalian, China). GAPDH and U6 were used as endogenous controls for normalizing the expression of genes and miRNAs. All experiments were performed with five biological replicates, and each sample was tested in triplicate. RT-qPCR was performed on a LightCycler 480II (Roche, Basel, Sweden) using SYBR Premix Ex Taq II. The cycling conditions were pre-denaturation at 95 °C for 5 s, then 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Melting curve analysis was performed, and the relative expression levels were determined using the 2 ^ΔΔCt method [33 (link)]. The p-value calculation was performed by t-test, and p < 0.05 was used to indicate significant differences. The primers were designed using Primer 5 (listed in Supplementary Table S1).