Cfx96 system

Total RNA was extracted from HK-2 cells grown under LG or HG utilizing Trizol reagent (Takara, Dalian, China) and transcribed into cDNA using PrimeScript RT Reagents (Takara) as per manufacturer guidelines. On a Bio-Rad CFX96 system, the TB Green Premix Ex Taq (Takara) was utilized to conduct quantitative real-time PCR (qRT-PCR) (Bio-Rad, CA, USA). The relative gene expression was determined utilizing 2–ΔΔCT and standardized by β-actin. Table S1 included all primer sequences.

Total RNA was obtained from the distal ileum using an RNAiso Plus kit (Takara, Japan). For cDNA synthesis, total RNA was reverse transcribed to cDNA with a PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) following the kit instructions. SYBR green-based RT – qPCR was performed on a Bio-Rad CFX96 system using a TB Green Premix Ex Taq II Kit (Takara, Japan). The stably expressed housekeeping gene hypoxanthine phosphoribosyltransferase 1 (Hprt1) was used to normalize mRNA expression, and relative expression was quantified using the ΔΔCT method in Microsoft Excel (Microsoft, USA). The specified primers were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) or available from previous literature (Macias-Ceja et al., 2019 (link); Wu et al., 2020 (link); Mills et al., 2021 (link); Sun et al., 2021 (link)) and Primer Bank (http://pga.mgh.harvard.edu/primerbank/index.html), and purchased from Sangon Company (Shanghai, China) with the detailed sequence information provided in Table 1.

Total RNA was extracted from root tissues using the SV Total RNA Isolation System kit (Promega, USA). For qPCR, total RNA was collected from samples with different time points after nitrate supply. The DNA was digested using RNase-free DNase I. The Prime ScriptTM RT Reagent kit (Takara, Dalian, China) was used to generate cDNA. The qPCR experiment was performed using a Bio-Rad CFX96 system with SYBR® Premix Ex Taq™ II (Takara, Dalian, China). The gene ZmUPF1 [59 (link)] was used as an internal control. For the RT-PCR experiment, the KOD polymerase (Toyobo) was used on testing AS events in the untreated and treated samples, respectively. The primers used for qPCR and RT-PCR were included in supplemental Table S8.

Total RNA was extracted from the frozen tissue using Tiangen’s RNA prep pure plant kit (Cat.DP432, Beijing, China) according to the manufacturer’s instructions. Next, 2 μg RNA was used for the first strand of cDNA synthesis using reverse transcriptase (Thermo Scientific, #EP0441). Real-time PCR amplification was carried out with the Bio-Rad CFX96 system using SYBR Green I (Takara, DRR081A, Dalian, China). PCR conditions were 3 min at 95°C followed by 40 cycles of the following: 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. The primer pairs used were FtCuAO 5'-ACCTCAGGTGAAGCAGTCAA-3' and 5'-GGGATTTCGCACCCTCATTC-3'; FtRPB1 5'-CTCACGACAACCACCATTCC-3' and 5'-CCTCCTTGTGTGGAGTGTCT-3'; and FtDHE1 5'-CAGAGGAGCTTGCTTGGTTG-3' and 5'-CGCAAATGGCAGACACTGAT-3'. The FtH3 gene was amplified as a reference gene, since its expression is unaffected by abiotic treatment (Li et al., 2019 (link)), using primers 5'-GAAATTCGCAAGTACCAGAAGAG-3' and 5'-CCAACAAGGTATGCCTCAGC-3'.

The TRIzol reagent (Invitrogen) was used to extract total RNA from cells. The RNA was transcribed to cDNA utilizing a Reverse Transcription Kit (Takara Biotechnology Ltd., Dalian, China). Next, a real-time qPCR was performed on a Bio-Rad CFX96 System using a SYBR Green Real-Time PCR Kit (Takara). The primer sequences were obtained from OriGene Technologies (Beijing, China) and listed in Table 1, where GAPDH and U6 were used as the internal controls for mRNAs and miRNA, respectively. Relative gene fold changes were measured using the 2^–ΔΔCt method.

By using the TRIzol reagent (Invitrogen, Carlsbad, USA), we supplemented the extraction of total RNA from CC cells, which were then reversely transcribed into cDNA by using the Reverse Transcription Kit (Invitrogen). RT-qPCR was processed on the Bio-Rad CFX96 system using the SYBR-Green Real-Time PCR Kit (Takara Bio Inc., Tokyo, Japan). The normalization was set as GAPDH or U6, with expression fold changes calculated by 2^−ΔΔCt methods. Each experiment went through three repeats.

Uterine tissue samples were thawed at low temperatures. Uteri samples and liquid nitrogen were added to mortar; the samples were ground into powder at low-temperature RNA, and protein was extracted. Total RNA from BEECs and endometrial tissues was isolated using RNAiso Plus (Takara, Maebashi, Japan), according to protocols from the manufacturer. Extracted RNA was quantified, and 1 μg of RNA was added to a genomic DNA elimination reaction for reverse transcription into a cDNA template with gDNA Eraser (Takara). Quantitative PCR was performed with a Bio-Rad CFX96 system using the SYBR Green Plus Reagent Kit (Takara). The reaction conditions were as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The mRNA expression levels were measured relative to the mRNA of the β-actin reference gene using the 2^−ΔΔCt method. The primer sequences for each gene are reported in Table 2.