Sybr green master mix

Total RNA was isolated from cells and reverse transcribed to cDNA using a high capacity cDNA reverse transcription kit (4368814, Applied Biosystems). Reactions were performed using 20ng of cDNA per condition combined with forward and reverse primers (Table S1), and SYBR green master mix (NEB: M3003) in a 10ul reaction volume. Assays were performed in the QuantStudio 5 Real-Time PCR system (A34322, Thermo Fisher Scientific) measuring binding of SYBR green to DNA, with ROX as a passive dye. Reaction conditions were as follows: 2 minutes at 50°C, 10 minutes at 95°C, and 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. Cycle threshold (CT) values were calculated using QuantStudio Design and Analysis Software v1.5.1 (Thermo Fisher Scientific). Relative gene expression was determined by the 2^−ΔΔCT method using the geometric mean expression of two validated endogenous control genes (ACTB and B2M) to ensure the reliability and reproducibility of observed effects (Table S3).

Tissues (around 100 mg) were homogenized in liquid nitrogen with a glass homogenizer. Total RNA was extracted using QIAzol^® Lysis Reagent (QIAGEN Science, Germantown, MD, USA) according to the manufacturer’s instructions. RNA was converted into cDNA using the RevertAid™ first-strand cDNA synthesis kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Next, we conducted a quantitative polymerase chain reaction using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). The reaction solution (20 μL) contained 10 μL of SYBR Green master mix (New England Biolabs, Ipswich, MA, USA), 4 μL of cDNA, and 6 μL of primer set (200 nmol/L). The PCR reactions were conducted as follows: 2 min at 50 °C, 10 min at 95 °C, and 40 cycles at 95 °C for 15 s, followed by 1 min at 60 °C. The relative expression levels were determined as the Δcycle threshold (ΔCt) [19 (link)]. All primer sets used in the present study are shown in Supplemental Table S1.

We utilized a CFX Connect qPCR detection instrument to confirm the sequencing findings for gene expression profiling. The elongation factor 1-alpha gene (accession number: AB061263) served as a reference gene, while four drought tolerance-related genes were randomly chosen (Table 1) for validating the RNA-Seq results. The NCBI Primer-BLAST program, a resource for generating gene-specific primers, was utilized to brand primers for evaluating relative gene expression. After that, validation analysis was performed in 96 well-plates (Applied Biosystems, Foster, California) with SYBR Green master mix (Luna^® Universal qPCR Master Mix, NEB) in qTower (Applied Biosystems) at 95°C for 60 seconds, proceeded by 40 cycles of 95°C for 15 seconds, and 58°C for 30 seconds. The gene expression level was performed by the comparative 2^−ΔΔ CT methods [29 (link)] using all samples in triplicate for normalization.

RNA from HMEG cell lines was isolated using the NucleoSpin® RNA Kit from Macherey & Nagel. RNA from paraffin embedded tissue was isolated using the RNeasy FFPE Kit from Qiagen Kit. RNA isolations were according to the manufacturer’s protocol. cDNA was synthesized from 1 µg RNA using random primers and Omniscript Reverse Transcription (RT) Kit (Qiagen). Quantitative real-time reverse transcription PCR was performed using SYBR Green Master Mix (NEB) on a 7500 Fast Realtime PCR system (Applied Biosystems). The ribosomal protein RPLP0 was used as housekeeping gene. All experiments were performed in duplicates and are displayed in ±SD. The primers were supplied by Biomers. The sequences are provided in Supplementary Table 1.

Total RNA was extracted from the colon tissues (50 to 100 mg) using TRIzol reagent (Thermo Fisher Scientific). The total RNA was quantified and then reverted to cDNA. Real-time PCR was measured using the SYBR green master mix (New England Biolabs [NEB]). Cycle threshold (C_T) was used to calculate the relative expression of the target gene by the 2^−ΔΔCT method. Primers for mRNA expression measurement are listed in Table S2.

RNA was extracted using the RNeasy Mini Kit (QIAGEN), and cDNA was produced with the First Strand cDNA Synthesis Kit (LunaScript RT SuperMix Kit; New England Biolabs). qPCR reactions were performed with SYBR Green Master Mix (Luna Universal qPCR Master Mix; New England Biolabs) and run on a CFX96 Real-Time PCR machine (Bio-Rad). Relative gene expression values were calculated with the –ΔCt method, using GAPDH as a housekeeping gene for normalisation. Oligonucleotides used for qPCR are provided in Table S16.

Table S16 List of primers used to quantify gene expression by qPCR and for ChIP–qPCR.