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36 protocols using EASYspin Plus Complex Plant RNA Kit

RNA extraction was performed using an EASYspin Plus Complex Plant RNA Kit (Aidlab, Beijing, China). We used 1% agarose gel electrophoresis to assess the integrity of the RNA. The bands were clear, and the brightness of 28S at about 1000 bp was about twice that of 18S at about 600 bp (Figure 1), indicating that the integrity of the RNA was good. The RNA samples were then quantified using NanoPhotometer P330 (Imlen, Germany). The A260/A280 of qualified RNA must be in the range of 1.8–2.2, and A260/A230 > 2.0. Only RNA that met the criteria was used for subsequent analyses. cDNA Synthesis SuperMix (TransGen, Beijing, China) was used to reverse-transcribe 400 ng of total RNA from each sample into cDNA.
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Genomic DNA was extracted from fresh leaves using the CTAB method [32 (link)]. The integrity of the DNA was assessed using electrophoresis on a 1% (w/v) agarose gel on a gel-imaging system (Syngene, Cambridge, UK). The concentration of DNA was adjusted to 50 ng/μL, as determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Total RNA was extracted from fresh tissues collected at different growth stages using the EASYspin Plus Complex Plant RNA Kit (Aidlab, Beijing, China). First-strand cDNA was synthesized with the AMV First-Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China).
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Total RNA was extracted from leaves using the EASYspin Plus Complex Plant RNA Kit (Aidlab Biotech, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized in a reverse transcription reaction including 1 μg of total RNA, oligo(dT) primer, and M-MLV reverse transcriptase (Promega, Madison, WI, USA). PCR cloning of full-length PeWRKY1 was performed in a total volume of 25 μl containing 1 μl of cDNA product, 1 U of Ex Taq polymerase (Takara, Dalian, China), 2.5 μl of 10× Ex Taq buffer, 2 μl of dNTP mixture (2.5 mM), and 0.5 μl of forward and reverse primers (10 μM) designed based on the homologous WRKY1 sequence from P. euphratica. The 5′ to 3′ primer sequences were: forward, 5′-ATGGCTGCTTCTTCAGGGAG-3′; and reverse, 5′-CTACCAAAAACTCTCTACTTCC-3′. The PCR product was gel purified and then ligated to the pMD18-T vector (Takara) for DNA sequencing.
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RNA was extracted with EASY spin plus complex plant RNA kit provided by Aidlab company (Beijing, China), reverse transcription RNA into a single-stranded cDNA by using the PrimeScriptTM RT reagent kit and gDNA Eraser kit provided by TaKaRa. The primers were designed by DNAMAN8.0 software and tested to ensure amplification of single discrete bands with no primer-dimers. Product size from 140bp to 200bp. Primer sequences are shown in detail in the Supplementary Table S1. Longan actin gene was used as the reference gene for expression analyzing [38 (link)]. The qRT-PCR experiment used CFX96 real-time PCR Detection system (Bio-Rad laboratories, Hercules, CA, USA) and TB Green mixture (TaKaRa Bio, Shiga, Japan). The relative expression of specific gene was quantitated with the 2−ΔΔCt calculation method [39 (link)].
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Total RNA was extracted using the EASY spin Plus Complex Plant RNA Kit (Aidlab, China), and then tested using the Nanodrop (Thermo Fisher Scientific, USA), Qubit 3.0 (Invitrogen, USA), and Agilent2100 (plant RNA Nano Chip, Agilent, USA) for purity, concentration, and integrity, respectively. After the removal of ribosome RNA by the Ribo-Zero Magnetic Kit (Epicenter, USA), the libraries were built using a TruSeq RNA Sample Prep Kit (Illumina, USA). An Illumina HiSeq X-ten platform (Illumina) set with length of 150-bp pair-end reads was then used for sequencing (Mega Genomics, China). Sequences of adaptor and low-quality trait were trimmed from raw reads, and the rest reads were mapped to the genome sequences of common tea (C. sinensis) (Wei et al., 2018 (link)), using the CLC Genomic Workbench 9.5 (Qiagen, USA). The reads with sequence similarities of >60% to the tea genome sequences were eliminated to reduce interference of the host background, and the remaining unique reads were de novo assembled using the Trinity program (Grabherr et al., 2013 (link)). The resulted contigs were subjected to BLASTx and BLASTn searches against viral (taxid:10239) and viroidal (taxid:2559587) sequences of local datasets retrieved from the National Center for Biotechnology Information (NCBI) databanks. These processes allowed the identification of the contigs with viral sequence attributes.
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Total RNA was extracted from the fourth leaf from bottom at six-leaf stage using the EASYspin Plus Complex Plant RNA Kit (Aidlab Biotech, Beijing, China). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) prior to library construction. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, San Diego, CA, USA) following manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. The cDNA libraries were sequenced on the Illumina sequencing platform by Metware Biotechnology Co., Ltd. (Wuhan, China).
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The RNA-seq and de novo assembly was performed as previously described [21 (link),22 (link),23 (link)]. Briefly, total RNA was extracted from the sampled leaf tissues using the EASYspin Plus Complex Plant RNA Kit (Aidlab Biotech, Beijing, China). Ribosomal RNAs were removed by the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). An RNA library was constructed using the TruSeq RNA Sample Prep Kit according to the manufacturer’s protocol (Illumina, San Diego, CA, USA), followed by sequencing on the Illumina HiSeq X-ten platform (Biomarker Technologies, Beijing, China). Low-quality reads were filtered and adapters of the paired-end raw reads were trimmed using the CLC Genomics Workbench 9.5 software (QIAGEN, Hilden, Germany). The clean reads were de novo assembled into contigs using the Trinity v2.3.2 program (Broad Institute and the Hebrew University of Jerusalem, Cambridge, Massachusetts and Jerusalem, USA and Israel) [22 (link)].
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Petal tissue at the early developmental stage (from <4 mm buds) was used for RNA-Seq. Total RNA was isolated using an EASYspin Plus Complex Plant RNA Kit according to the manufacturer's recommendations (Aidlab, Beijing, China). Three biological replicates were performed. RNA-Seq libraries were constructed according to the user manual and sequenced to produce 150-bp paired-end reads on BGISEQ-500 platform. Clean reads were obtained by removing sequences containing adapters and poly-N runs and low-quality reads. The clean sequence tags were mapped to a TM-1 genome (JGI v1.1 annotation, https://phytozome.jgi.doe.gov). The fragments per kilobase of exon per million reads mapped (FPKM) were calculated to indicate the expression level using cufflink (Trapnell et al., 2010 (link)). Pairwise comparisons (CS-B18 versus TM-1 and YangOB versus TM-1) were conducted to find differentially expressed genes by using the R program DEGseq software with the parameters: Q-value ≤ 0.001 and log2 (fold change) ≥ 1 (Wang et al., 2010 (link)). The Gene Ontology (GO) terms and KEGG enrichment analysis were conducted using the R program phyper (FDR ≤ 0.01). Sequencing data were deposited in the NCBI SRA database (BioProject accession number: PRJNA541186).
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Total RNA was isolated from the stem phloem samples using an EASYspin Plus Complex Plant RNA Kit (Aidlab, Beijing, China), from the inoculated N. benthamiana leaves using a TransZol Plant RNA Kit (TransGen Biotech, Beijing, China), and from L. theobromae mycelia using TRIzol reagent (Invitrogen). We used 2 µg of each total RNA sample to synthesize cDNA using a Superscript III First-Strand Synthesis SuperMix Kit (Invitrogen). The qRT-PCRs were run on a 7500 Real-Time System (Applied Biosystems) following the manufacturer’s protocols. Relative gene expression levels were calculated using the comparative 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primers used are listed in Supplementary Table S1.
Defense suppression tests were performed as previously reported (Chen et al., 2015 (link)). Briefly, 10-day-old seedlings of N. benthamiana were treated with 1 µM flg22 (Sangon Biotech, China). The expression of the PTI-associated genes NbACRE31, NbGRAS2, and NbPTI5 were determined by qRT-PCR. In addition, qRT-PCR was also used to determine the expression levels of the defense-related genes in the SA and jasmonic acid (JA) signaling pathways, including PATHOGENESIS RELATED PROTEIN 1 (NbPR1) and LINOLEATE 9S-LIPOXYGENASE 5 (NbLOX), which are specifically induced by SA signaling. The NbEF1α and NbTUBULIN6 genes were used as internal controls.
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The total RNA of each species was extracted from one plant using the EASY spin Plus Complex Plant RNA Kit (Aidlab, Beijing, China). The RNA purity, concentration, and integrity were evaluated using a Nanodrop (Thermo Fisher Scientific, Cleveland, OH, USA), Qubit 3.0 (Invitrogen, Waltham, MA, USA), and Agilent2100 (plant RNA Nano Chip, Agilent, Santa Clara, CA, USA), respectively. The ribosome RNA was depleted by the RiboZero Magnetic Kit (Epicenter, Madison, WI, USA), and a library was then built using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, USA). Furthermore, RNA-seq was conducted by Beijing Genomics Institution (BGI) using the BGI500 platform set 100 bp for the length of paired-end (PE) reads, by Mega Genomics (MG, Beijing, China) using an Illumina HiSeq X-Ten platform (PE 150 bp), or by Berry Genomics Corporation (BGC, Beijing, China) with an Illumina NovaSeq 6000 platform (PE 150 bp).
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