Qtower3g touch

For the expression analysis of candidate genes, we harvested leaf and root tissue from 16 week old carrot plants (cola and yellow mutants). The root samples are heterogeneous samples including cortex, xylem and phloem tissues. The leaf samples comprise of young leaf and petiole tissues. To reduce plant-specific differences, each sample was pooled from three individual plants, frozen in liquid nitrogen and ground to a fine powder. Total RNA was extracted from 100 mg tissue powder using the innuPREP Plant RNA Kit (Analytik Jena, Jena, Germany) according to the manufacturer’s instructions. RNA integrity was confirmed by agarose gel electrophoresis. Random hexamers were used as primers for first-strand cDNA synthesis with 2 µg total RNA as a template and Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, USA). Carrot tubulin 3α (DcTUB), heat shock 70 (DcHSP) and protein phosphatase 2 (DcPP2A) were used as internal reference genes. The expression levels of DcTPS04, DcTPS26, DcTPS27, DcTPS54, DcTPS55, DcTUB, DcHSP and DcPP2A genes were analysed with the qTOWER³ G touch (Analytic Jena, Jena, Germany) equipment and gene-specific primers (Tab. S4). The results were calculated using the Pfaffl method^{59 (link)}.

Total RNA was extracted from the shoot and root tissues using Sepasol-RNA I (Nacalai Tesque), and reverse transcription was conducted using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Maebashi, Japan). Quantitative PCR was conducted using the KAPA SYBR FAST qPCR Master Mix 2× (Kapa Biosystems, Boston, MA, USA) and qTOWER3G touch (Analytik Jena AG, Germany). Relative mRNA abundance was determined using the ΔΔCt method, and ubiquitin (UBQ2, accession no. J05508) was used as a constitutive internal control. Gene specific primers for quantitative PCR are listed in Supplemental Table S3 [48 (link),49 (link),50 (link)].

OTULIN^WT, OTULIN^M86I, and OTULIN^W167S were subjected to thermal denaturation in a real‐time thermal cycler (qTOWER³ G touch, Analytik Jena) using emission and excitation wavelengths of 490 and 580 nm, respectively. Quintuplicates were measured in 2× SYPRO^®‐Orange (SA) at a protein concentration of 4 µM in gel filtration buffer. Experiments were performed in white 96‐well qPCR plates (Sarstedt), which were sealed with adhesive qPCR seal (Sarstedt) and centrifuged at 1,000 g for 1 min and 20°C to remove air bubbles. A temperature gradient from 30 to 90°C was applied with a ramping rate of 1°C/s and a ΔT of 1°C while fluorescence was detected after an equilibration time of 30 s. Curves were referenced against buffer controls and T_m values calculated and averaged from the derivatives of the melting curves (‐dRn/dT) as implemented in qPCRsoft 4.0 (Jena Analytik).

For analysis of the aptamer libraries, quantitative PCR (qPCR) was performed with the eluates of the respective SELEX rounds using the qTOWER³G touch (Analytik Jena, Jena, Germany) and the fluorescent dye SYBR Green I (Sigma-Aldrich, St. Louis, MO, USA) under the following thermal cycling conditions: an initial step of 3 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 56 °C, and 10 s at 72 °C. A master mix was prepared for each reaction batch. Therefore, a 5 × Herculase II Reactions buffer (Agilent Technologies, Inc., Santa Clara, CA, USA), a Herculase II Fusion DNA Polymerase (Agilent Technologies, Inc., Santa Clara, CA, USA), and a 100-mM dNTP mix (Agilent Technologies, Inc., Santa Clara, CA, USA) were used. Also, 100-mM unmodified primers (forward primer: 5′-TAG GGA AGA GAA GGA CAT ATG AT-3′; reverse primer: 5′-TCA AGT GGT CAT GTA CTA GTC AA-3′) (biomers.net GmbH, Ulm, Germany) were used. For each assay, 0.003761 pmol of the respective aptamer DNA was employed. All amplification reactions were performed in triplicates, including no-template controls (NTC) in each run to check for impurities.

To obtain high-quality clean data for downstream analyses, raw reads were screened to exclude those with sequencing adapters, low quantity reads and reads containing ploy-N. The remaining clean reads were then mapped to the reference full-length transcriptome sequence using STAR. Differential expression analysis was conducted using the DESeq R package (1.10.1), and genes with FDR < 0.05 and |log2FC| > 1 were considered differentially expressed. Gene ontology (GO) enrichment analysis of the differentially expressed genes was performed through the GOseq R package [34 (link)]. In vivo metabolic analysis was carried out using the KEGG database (Kyoto Encyclopedia of Genes and Genomes, http://www.kegg.jp), accessed on 20 September 2022.
Eight differentially expressed genes were selected for validation through quantitative real-time PCR (qRT-PCR). The cDNA were reverse transcribed using FastKing gDNA Dispelling RT SuperMix kit (Tiangen, Beijing, China). The primer pairs were designed by Primer Premier 5 and are listed in supplemental Table S12. The qRT-PCR reaction was carried out using 2× HQ SYBR qPCR Mix (Zoman, Beijing, China). Each sample was run in triplicate on a fluorescence quantitative instrument (qTOWER3Gtouch, Germany, Analytik Jena). The relative expression of genes was quantified using the 2^−ΔΔCT method.

Age-synchronized nematodes were prepared as described above. Worms were grown on S medium with OP50 and OP50 (S⁺) bacteria. Three biological replicates of animals at 4 days and 12 days after the L4 stage were used for the RNA isolation. The first-strand cDNA synthesis was then performed from the total RNA with the Maxima H Minus M-MuLV reverse transcriptase kit (Sangon Biotech, China). Expression levels of genes were measured with internal control of bmp-3. Real-time PCR was carried out by the 2 × SG Fast qPCR Master Mix Kit (Sangon Biotech, China) on Analytikjena & qTOWER3G touch (Analytikjena, Germany). Three independent trials were performed, and the relative mRNA quantification of the desired genes was analyzed by the comparative CT (ΔΔ CT) method^{46 (link)}. The qRT-PCR primers were listed in Supplementary Table 20.

Pathogen-infected and control plant leaves were harvested at 24 h posttreatment for RNA isolation by using a Plant RNA Extraction Kit (Omega Bio-Tek, USA). First-strand cDNA was generated using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (TOYOBO, Japan). Quantitative real-time PCR was performed on a qTOWER³G touch (Analytikjena, Germany) with KOD SYBR qPCR Mix (TOYOBO). The PCR program was performed as described in a previous study (Yang et al. 2019 (link)). The gene expression levels relative to those of the rice OsActin (LOC_Os03g50890) gene were analysed using the 2^-△△Ct analysis method. The OsActin gene was used as an internal control to standardize the results. Gene expression levels were analysed by qRT-PCR assays, which were repeated at least twice in triplicate.