Hipure plant rna kit

Total RNA was isolated with the HiPure Plant RNA Kits (Magen) according to the manufacturer’s instructions. After concentration detection by ultraviolet spectrophotometer, first-strand cDNA was synthesized by using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) from 1 μg total RNA according to the manufacturer’s instructions. Real time RT-PCR was conducted using a model 7500 real-time PCR system (Applied Biosystems). To normalize the amount of total cDNA present in each reaction, the tobacco L25 ribosomal protein (NCBI accession number: L18908) gene was used as an internal control according to Schmidt and Delaney [14 (link)]. Real-time RT-PCR was performed in a total volume of 20 μl reaction mixtures with gene-specific primers (S2 Table). Each reaction included 10 μl of SYBR green real-time PCR master mix (Bio labs), 0.5 μM (each) forward and reverse primers, and 2 μl of cDNA template (equivalent to 100 ng cDNA). The amplification was performed using the following cycle parameters: 94°C for 30 s, followed by 40 cycles of 94°C for 15 s, and 60°C for 40 s for plate reading. Three independent biological repeats were performed for each treatment. Gene expression levels were calculated from the threshold cycle (CT) according to the 2^−ΔΔCT method.

To validate the relative expression of four sgRNAs of both the CRISPR/Cas9 and the PTG/Cas9 systems in kiwifruit, we designed four pair primers (sgRNA1‐F/sgRNA‐R, sgRNA2‐F/sgRNA‐R, sgRNA3‐F/sgRNA‐R and sgRNA4‐F/sgRNA‐R) using the online software Primer3Plus, which were then synthesized commercially (Sangon Biotech Co., Ltd., Shanghai, China) (Table S1). RNA was extracted from callus lines transformed with A1/A2/B1/B2 using HiPure plant RNA kits (Magen, Guangzhou, China). The cDNA was prepared from all samples using a one‐step gDNA removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China) and was then used as the input for quantitative PCR (qPCR) experiments. Each qPCR was performed in a total volume of 20 μL, containing 10 μL of Tip Green qPCR SuperMix (TransGen, Beijing, China), 0.2 μm of each primer, 1 μL of 1 : 5 diluted cDNA and 8.2 μL ddH₂O. Thermal cycling consisted of a hold at 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. The temperature was then gradually raised by 0.5 °C every 10 s for performing melting curve analysis. Each sample was amplified in triplicate, and all qPCRs were performed on a LightCycler 480 system (Roche, Basel, Switzerland). The ΔΔCt method was employed with Achn107181 (kiwifruit actin gene) and Achn381211 (protein phosphatase 2A, PP2A‐like gene) as endogenous controls (Petriccione et al., 2015).

Total RNA was isolated from C. rosea and A. thaliana using HiPure Plant RNA Kits (Magen, Guangzhou, China), and the cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Quantitative reverse transcription (qRT)-PCR was conducted using a LightCycler^® 480 Gene Scanning system (Roche, Switzerland) and TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). Gene expression levels were normalized using the C. rosea reference gene CrEF-α as internal control. The primer pairs (CrPIP2;3RTF/CrPIP2;3RTR and CrEF-αRTF/CrEF-αRTR) used for qRT-PCR are listed in Table S3.

The kiwifruit callus was induced from Actinidia chinensis cv. Hongyang as previously described [42 ,43 (link)]. In the light and temperature treatment experiment, the callus was incubated under the following conditions: 16 °C /light (intensity of 80 μmol m⁻² s⁻¹), 24 °C/light, 32 °C/light, 16 °C/dark, 24 °C/dark, and 32 °C/dark. The callus was sampled at 0, 4, 8, 12, 16, 20, and 24 days after treatment.
In the VIGS experiment, Agrobacterium was injected into the fruit of ‘Hongshi No. 2′. In the transient expression experiment, Agrobacterium was injected into the fruit of A. arguta cv. Baobeihong, a purple A. arguta var. purpurea cultivar. The samples of ‘Hongshi No. 2′ and ‘Baobeihong’ were collected from a germplasm garden at Wufeng mountain, Enshi city, Hubei province.
The genomic DNA used in this study was extracted from ‘Hongyang’ leaves using a DNA extraction kit (DN38, Aidlab, Beijing). Total RNA was extracted using HiPure Plant RNA kits (R4151-03, Magen, Shanghai). The cDNA libraries were constructed by using HiScript II QRT SuperMix (R223-01, Vazyme, Nanjing).

Total RNA of six sample were acquired using HiPure Plant RNA Kits (Magen, China). We verified and purified the RNA quality by NanoDrop ND-2000 spectrophotometer (Nano-Drop, Wilmington, DE) and Dynabeads® Oligo (dT) 25 (Life Technologies, USA), respectively. After that, a cDNA library was generated using the purified mRNA, which was used for cluster generation using the TruseqTM RNA sample prep Kit and were subsequently sequenced using the HiSeq 4000 SBS Kit.
To obtain clean reads, the SeqPrep and Sickle software were used to filter the raw data. The clean reads were assembled using Trinity software to generate the unigenes. To identify potential protein-coding genes and annotate the function of these unigenes, all assembled sequences were screened for ORF prediction using EMBOSS software. The predicted protein-coding sequences were searched in the Nr (NCBI non-redundant) database, Pfam database, KEGG (Kyoto Encyclopedia of Genes and Genomes pathway) database, String database, GO (gene ontology) database and Swiss-Prot database using BlastX (E value <1e-5). The proteins with the highest matching value were considered to contain annotation information.

Total RNA from grape berry and transgenic tomato tissues was isolated using HiPure Plant RNA Kits (Magen, China). Then, cDNA was synthesized using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO, China). To normalize the cDNA samples, SlEF-1α, SlGADPH, and SlActin from tomatoes were selected as internal controls. Gene-specific primers (Supplementary Table S1) were designed, and qRT-PCR was performed using an UltraSYBR Mixture Kit (CWBIO, China) with a Rotor-Gene^® SYBR^® Green PCR Kit (QIAGEN, Germany). To determine the different concentrations of cDNA, the threshold cycle for each qPCR was identified and compared against the internal standards (SlEF-1α, SlGADPH, and SlActin for tomatoes).

The roots, rhizomes and the main aerial tissues of leaflets and petioles of C. chinensis were used for RNA-sequencing (Supplementary Fig. 1). The total RNAs of these tissues were extracted using a HiPure Plant RNA Kit (Magen, Guangzhou, China) and further purified using a NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Inc.). Approximately, 2 μg of RNA for each sample was prepared to construct the RNA-seq libraries using an Illumina TruSeq library Stranded mRNA Prep Kit, after which the RNA was sequenced on the Illumina platform.