Hipure plant rna mini kit

Three long SL accessions and three short SL accessions were selected for RNA-sequencing based on haplotype analysis. Seeds were germinated in a petri dish in a growth chamber. RNA was extracted from five-day-old seedlings using the HiPure Plant RNA Mini Kit (Magen, Guangzhou, China) with three biological RNA replicates. RNA-Seq was conducted by Annoroad Gene Technology Co., Ltd. (Beijing, China). RNA samples were sequenced using an Illumina HiSeq-2,500, producing 10 Gb of raw sequencing data. Data analysis was conducted using HISAT2-Stringtie-Deseq2 pipeline (Pertea et al., 2016 (link)). Raw counts of each sample exported from Stringtie were imported and normalized by DEseq2 (Love et al., 2014 (link)). Genes with average read counts less than 10 in all samples were filtered out for further analysis. For each accession, transcript abundance was calculated as the mean normalized counts of three biological replicates. p values between long and short SL accessions were estimated by the Student’s t-test. The genes with corrected p values (false discovery rate or q-value) ≤0.05 were identified the differentially expressed genes. GO enrichment analysis was performed using DAVID (Sherman et al., 2022 (link)).

Total RNA was extracted from each sample using a HiPure Plant RNA Mini Kit (Magen, Guangzhou, China). First Strand cDNA Synthesis Kit (Toyobo, Kita-ku, Osaka, Japan) with random primers was used to synthesize first-strand cDNA, and 1 µg total RNA was added per 20 µL reaction volume, as previously described (He et al., 2020 (link)). Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) and ABI 7900HT sequence detection system (Applied Biosystems QuantStudio 5, Foster City, CA, USA) were used to perform quantitative PCR. The 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to quantify the relative gene expression. In addition, the expression of T. aestivum cell division cycle (TaCDC) gene (Accession Number: XM_020313450) was analyzed and used as an internal control (Zhang et al., 2019 (link)). Each treatment in this study had at least three biological and three technical replicates. All the gene-specific primers used in this study were listed in Supplementary Table S1.

Total RNA was extracted from each wheat sample using a Hipure Plant RNA Mini Kit (Magen, Guangzhou, China), according to the manufacturer’s instructions, and stored at −80 °C until use. The first-strand cDNA was synthesized from 1 μg total RNA per 20 μL reaction volume using a First Strand cDNA Synthesis Kit (Toyobo, Kita-ku, Osaka, Japan), according to the manufacturer’s instructions, and as previously described [37 (link)]. The real-time quantitative polymerase chain reaction (RT-qPCR) assay was conducted on an ABI7900HT Sequence Detection System (Applied Biosystems QuantStudio 5, Foster City, CA, USA) using the Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). At least 3 biological replicates, with 3 technical replicates, were used for each treatment. The relative gene expression levels were calculated according to the 2^−ΔΔC(t) method [38 (link)]. In each reaction, the Triticum aestivum cell division cycle (CDC) gene was used as an internal reference gene [35 (link)]. All primers used in RT-qPCR are listed in Table S1.

Translation efficiency was assayed as previously described with minor modifications (Merchante et al., 2015 (link)). First, 10 g of N. benthamiana leaves was ground into fine powder in liquid nitrogen. Then, 2 g of sample was used to extract the total RNA with a HiPure plant RNA mini kit (Magen). Next, the other 8 g of sample was suspended in 20 ml of polysome extraction buffer (200 mM Tris‐HCl, pH 9.0, 35 mM MgCl₂, 200 mM KCl, 25 mM EGTA, 1% vol/vol Triton X‐100, 1% vol/vol IGEPAL CA‐630, 5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], 50 μg/ml chloramphenicol, 100 μg/ml cycloheximide) at 4 °C for 20 min with slight shaking. The mixture was centrifuged twice at 16,000 × g for 20 min at 4 °C. Then, 16 ml of supernatant was slowly transferred onto 15 ml of sucrose buffer (1.75 M sucrose, 400 mM Tris‐HCl, pH 9.0, 35 mM MgCl₂, 5 mM EGTA, 200 mM KCl, 5 mM DTT, 50 μg/ml chloramphenicol, 50 μg/ml cycloheximide). After centrifugation at 200,000 × g for 4 hr at 4 °C, the supernatant was carefully removed and the polysomes in the bottom were resuspended in 300 μl of diethylpyrocarbonate [DEPC]‐treated water. The polysomes RNA was isolated using TRIzol reagent (Life Technologies) and then subjected to reverse transcription and real‐time PCR analysis.

Total RNA was extracted using the Hipure plant RNA Mini Kit (Magen, China). The RNA was then reverse transcribed into cDNA using the Primescript RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, China) according to the manufacturer's instructions. TB Green Premix Ex Taq II (Takara, China) was used for RT-qPCR analysis. The following PCR program on a CFX Connect real-time PCR detection system (Bio-Rad, USA) was used: 95 °C for 2 min, 45 cycles of 95 °C for 10 s, and 60 °C for 15 s, followed by a melting curve program. The rice Ubiquitin (UBQ) gene was used as an internal control. Relative expression levels of genes were calculated using the 2^−△△CT method (Livak and Schmittgen 2001 (link)). Primers used for RT-qPCR are listed in Supplemental Data Set 5.

The total RNA of the N. benthamiana tissue samples was extracted using the HiPure Plant RNA Mini Kit (Magen, Guangzhou, China). First-strand cDNA was synthesized using the First-Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The qRT-PCR was carried out using Hieff^® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) on an Applied Biosystems Quantstudio 6 Flex system (Applied Biosystems, Foster City, CA, USA), and the relative expression levels of the assayed genes were calculated using the 2^−ΔΔCt method. Each treatment had three biological replicates and three technical replicates. The primer sequences used in this study are listed in Table S3.

Total RNAs of 8-day-old seedlings were extracted using the Magen HiPure Plant RNA Mini Kit. A 2-μg quantity of the RNAs was reversely transcribed to cDNA using M-MLV reverse transcriptase (Takara). qPCR analyses were carried out using EvaGreen 2 × qPCR MasterMix (ABM) on a Bio-Rad CFX96 real-time PCR detection system. ACTIN 2 (At3g18780) mRNA was used as an internal control, and the relative expression level of each gene was calculated by the 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). The primers used for qPCR analysis are listed in Supplementary Table 3.