Nanovue plus

For qRT-PCR experiments, the RNAs of MSSA 6538 cells were isolated using the following procedure. MSSA 6538 cells (10⁷ cells/mL) were inoculated into 25 mL of LB medium at 37 °C in 250 mL shake flasks with overnight cultures (1: 100 dilution) and cultured for 3 h with shaking at 250 rpm. TBHQ (1 µg/mL) was then added, and incubation was continued for a further 4 h. Before sample collection, RNase inhibitor (Ambion, TX, USA) was added, and cells were centrifuged at 10,000 rpm for 2 min. Cell pellets were immediately frozen in dry ice and stored at −80 °C. RNA was isolated using the Qiagen RNeasy mini Kit (Valencia, CA, USA). RNA quality was assessed using a NanoVue Plus (Biochrom Ltd., Cambridge, UK).

Total RNA was extracted from T cells with TRIzol Reagent (TaKaRa) according to the manufacturer’s protocol (Yin et al., 2022a (link)). Chloroform (0.2 mL) was added to 1 mL of cell lysate and mixed by shaking. The mixture was then placed on ice for 15 min, centrifuged, and the uppermost clear phase was collected. An equal volume of isopropyl alcohol was then added to the mixture and the mixture was allowed to stand for 10 min before a second centrifugation. The liquid in the tube was discarded, and 0.6 mL of pre-cooled 75% (v/v) ethanol was added to wash the RNA precipitates on the tube wall. This process was repeated twice, after which the ethanol was discarded. When the edges of the RNA precipitate became clear, it was dissolved in 20 μL DEPC water and the concentration and purity of the dissolved precipitate were measured using a Biochrom NanoVue Plus. All steps were carried out on ice. Subsequently, 1000 ng of total RNA was reverse-transcripted to cDNA using Takara PrimeScript RT reagent kits (TaKaRa), which was then used for RT-qPCR on a LightCycler96 Instrument (Roche). The primer sequences are listed in Table 2, and ACTB/Actb was used as the internal mRNA control. Experiments were repeated in triplicate, and the relative RNA expression rates were calculated using the 2^-△△Ct method.

The specific quantitative RT-PCR primers for the selected GmRALFs were designed by Sangon Biotech (Shanghai, China). The total RNA was extracted by using the RNA prep pure plant kit (Tsingke, Beijing, China) from the frozen samples. All RNA was analyzed by electrophoresis and then quantified with a Nano Vue Plus (Biochrom, Harvard Bioscience Company, UK). The Mighty Script Plus First Strand cDNA Synthesis Master Mix (gDNA digester) (Sangon Biotech, Shanghai, China) was adopted to remove the genomic DNA and convert the total RNA to cDNA. The SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) was adopted to conduct the quantitative RT-PCR assay on a QuantStudio™ 6 Flex real-time system (Thermo Fisher Scientific, USA). Triplicate quantitative assays were performed on each cDNA sample. The soybean GmActin gene was used as internal control in reactions. The 2^-△△CT comparative CT method was used to estimate the relative expression level of genes. Students’ t-test was used for statistical analysis. Information on the qRT-PCR primer sequences can be found in Supplementary Table 1.

All samples were submitted to total DNA extraction using Quick-DNA™ HMW MagBead Kit (D6060; Zymo Research, Irving, USA) according to the manufacturer’s recommendation. We used NanoVue™ Plus (28956058; Biochrom™, Holliston, USA) to obtain quantitative and qualitative data from our DNA samples. Metagenomic libraries were prepared using Rapid Barcoding Kit (SQK-RBK004; Oxford Nanopore Technologies, Oxford, UK) with a DNA input of 400 ng per sample. All the steps were performed following the manufacturer’s recommendations. For each run that was performed during 24 h, we used 12 samples with different barcodes each. All flow cells used were FLO-MIN106D (R9.4.1; Oxford Nanopore Technologies, Oxford, UK) model, and we used a MinION sequencing device (Oxford Nanopore Technologies, Oxford, UK).

Frozen fresh gastric carcinoma (GC) and paired surgical margin (SM) tissue samples were collected from 156 patients in the WGS study (14 (link)). These samples were frozen in liquid nitrogen approximately 30 min after surgical dissection and then stored in a -80°C freezer for 2-5 yrs. Clinicopathological information was also obtained. The 2010 UICC tumor-node-metastasis (TNM) system was used to classify these GCs (15 ). Genomic DNA was extracted from these samples with a phenol/chloroform method coupled with RNase treatment. Concentrations of these DNA samples were determined with NanoVue Plus (Biochrom LTD, Cambridge, UK). DNA samples with OD_260nm/OD_280nm ratios ranging from 1.7 to 1.9 were used for the detection of gene copy number as described below.

The process used to extract genomic DNA followed the standard phenol-chloroform extraction method^{56 (link)}, which was then used as a template for subsequent analysis after quantification.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then quantified by NanoVue Plus (BiochRom LTD, Cambridge, England). The first-strand cDNA was synthesized using a PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, City, Country). A total of 800 ng of total RNA from each sample was reverse transcribed into first-strand cDNA and used as the template for qRT-PCR.

Total RNA was extracted from mononuclear cells using a RIBO-zol-A kit (AmpliSens, Bratislava, Slovak Republic). The RNA concentration and purity were measured spectrophotometrically on a NanoVue Plus instrument (Biochrom, Cambridge, England). An aliquot of 100 ng of mRNA was used as a template to generate cDNA by a REVERTA-L reverse transcriptase kit (AmpliSens, Bratislava, Slovak Republic). The cDNA samples were then diluted 1:25 prior to the PCR reaction. Quantitative cDNA amplification was performed with 5 μl of the diluted cDNA template in a total volume of 20 μl in a DT-322 real-time PCR cycler (DNA Technology, Moscow, Russia) using SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). The primer sequences (Sigma-Aldrich, Rehovot, Israel) were as follows. VDR [NCBI Gene ID 7421] forward primer (5’-GACCTGTGGCAACCAAGACT-3’), reverse primer (5’-AATCAGCTCCAGGCTGTGTC-3’); RPLP0 (reference gene) (NCBI Gene ID 6175) forward primer (5’-AGATGCAGCAGATCCGCAT-3’), reverse primer (5’-GTGGTGATACCTAAAGCCTG-3’). Standard cycling conditions were: 3 min initial enzyme activation at 94 °C then 42 cycles as follows—5 s at 94 °C and 30 s at the annealing temperature (60 °C for both VDR and RPLP0). The calibrator was the cDNA sample of a healthy volunteer with a high plasma 25(OH)D level. The relative expression level was quantified using the 2^-ΔΔCt method [20 (link)].