Augegreen qpcr master mix

Seven genes were randomly selected from the RNA-seq results. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank accession number: KU521365.1) gene served as the reference gene. The reliability of RNA-seq results was verified by RT-qPCR. Primers were designed using Primer Premier 6.0 software. The primer information is presented in Supplementary Table 1. The HiScript III RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used for reverse transcription of the samples after sequencing. RT-qPCR analyses were conducted using AugeGreen qPCR Master Mix (US Everbright Inc., Suzhou, China) and the Agilent Technologies Stratagene Mx3005P Real-Time PCR instrument (Palo Alto, CA, United States) according to Ren et al. (2020) (link). Each sample was repeated three times. Relative expression was calculated by the 2^–ΔΔCT method.

Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient mixture (DMEM/F12) and foetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY), hydrocortisone from Sigma–Aldrich (MO, USA), de Man Rogosa Sharpe (MRS) broth and Luria-Bertani (LB) broth from Aobox (Beijing, China), cell counting kit-8 (CCK-8), RIPA lysis buffer, BCA protein assay kit and BCIP/NBT colour development kit from Solarbio (Beijing, China), and ultrapure RNA extraction kit from CWBIO (Beijing, China). Uelris Il RT–PCR System for First-Strand cDNA Synthesis and AugeGreen™ qPCR Master Mix from US Everbright Inc. (CA, USA), and an Annexin V-FITC Apoptosis Detection Kit from Beyotime (Shanghai, China). Primary antibodies against p38 (Catalog #8690 T), phospho-p38 (Catalog #4511 T), ERK (Catalog #4695 T), phospho-ERK (Catalog #4370 T), JNK (Catalog #9252 T), phospho-JNK (Catalog #4668 T) and IκBα (Catalog #4812S) were acquired from Cell Signaling Technology (Danvers, MA, USA), and antibodies against NF-κB p65 (Catalog #bs-0465R), NF-κB phospho-p65 (Catalog #bs-0982R), phospho-IκBα (Catalog #bsm-52169R) and β-actin (Catalog #bs-0061R) were purchased from Bioss (Woburn, MA, USA).

Total RNAs were extracted from the salt stress-treated samples using the Omini Plant RNA Kit (CWBIO, Beijing, China). RNA was reverse transcribed into cDNA using a SuperRT cDNA Synthesis Kit (CWBIO, Beijing, China). Then, the cDNA was diluted 1:20 with ddH2O to be used as a template for quantitative real-time PCR (qRT-PCR). Then, qRT-PCR was performed using an AugeGreen™ qPCR Master Mix (US EVERBRIGHT, Suzhou, China) and samples were run in a LightCycler 96 System (Roche, CA, United States) using the following protocol: predenaturation at 95°C for 2 min; 45 cycles of denaturation at 95°C for 15 s and renaturation at 60°C for 60 s; and extension at 72°C for 30 s. The β-actin gene (X79378) was used as an internal reference gene. The primer pairs for qRT-PCR were summarized in Table S5. The relative expression levels were calculated using the 2^-ΔΔCt method, and three biological replicates and three technical replicates were performed for each sample.