Cfx manager software 2

Total RNA was extracted using an RNeasy Plus Micro Kit (QIAGEN, 74034) according to the manufacturer’s instructions^{15 (link)}. Cell lysates were spun using the kit’s gDNA Eliminator spin columns to remove genomic DNA. Total RNA was purified using RNeasy MinElute spin columns. First-strand cDNA was then synthesized from 1 µg of RNA using PrimeScript RT Master Mix (Takara, RR036A). Then 20 µl reactions were prepared by combining 4 µl of PrimeScript RT Master reaction mix, 2 µl of gene-specific enhancer solution, 1 µl of reverse transcriptase, 1 µl of gene-specific assay pool (20 ×, 2 µM), and 12 µl of RNA diluted in RNase-, DNase-, and genomic DNA-free water. We performed qPCR using TB Green Premix Ex Taq II (Takara, RR820Q) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad). Analysis was performed using Bio-Rad CFX Manager software 2.0. The data were normalized to RNA GAPDH and the fold change was calculated via the 2^−DDCt method. The relative concentrations of mRNA were expressed in arbitrary units based on the untreated group, which was assigned a value of 1.

Real-time PCR was performed in 15 μl reaction volume mixtures with 1 μl of cDNA, 7.5 μl of SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) and 2.5 mM of each primer (GPgV504-F and GPgV588-R). The following thermal protocol was used: 98 °C for 2 min; 45 cycles of denaturation at 98 °C for 5 s and annealing/extension at 60 °C for 5 s; final denaturation at 95 °C for 1 min and final extension at 65 °C for 1 min. Every plate included a non-template and a positive (cDNA from GPGV-infected plant) control. For each sample, three technical replicates were performed.
All reactions were performed on a CFX96 real-time system (Bio-Rad, Hercules, CA, USA) and amplification data were analyzed using the CFX Manager Software 2.0 (Bio-Rad). To allow comparability between assays, the baseline threshold was always set to 100 RFU (relative fluorescence units) and samples were considered positive for GPGV when threshold cycle (Ct) values were < 35, with values among 30 and 34 considered as low positive (Vončina et al. 2017 (link)). To compare different Ct values among samples with different symptom severity, statistical analyses were performed with the InStat GraphPad software package (La Jolla, CA, USA) using one-way ANOVA and Tukey-Kramer multiple comparisons test as post hoc test. A P value < 0.005 was considered statistically significant.

A. thaliana rosettes from Col-0, Bla-2 and Kon accessions were sampled after different times of mite infestation (1, 3, 6, 12 and 24 h). A. thaliana Col-0 flowers, roots, siliques, leaves from stems, and 1, 2 and 3 week-old rosettes were also collected. In addition, Arabidopsis entire plants from selected T-DNA insertion lines and from the non-transformed Col-0 controls were collected. Total RNA was extracted as previously described^{49 (link)} and reverse transcribed using Revert AidTM H Minus First Strand cDNA Synthesis Kit (Fermentas). RT-qPCR was performed for triplicate samples as previously described^{30 (link)} using a SYBR Green detection system (Roche) and the CFX Manager Software 2.0 (Bio-Rad). Ubiquitin was used as housekeeping gene for normalization. Gene expression was referred as relative expression levels (2^−dCt) or fold change (2^−ddCt)^{50 (link)}. Specific primers were designed through the PRIMER 3 program (http://bioinfo.ut.ee/primer3-0.4.0/). Primer sequences are indicated in Supplementary Table S2.

Single-cell suspensions of bone marrow (BM) cells (in PBS containing 2% FCS and 2 mM of EDTA) were stained with biotin-conjugated anti-Ly6G (1A8) and anti-CD19 (6D5) antibodies (BioLegend, San Diego, CA, USA) before removing positive cells with MagniSort™ streptavidin-negative selection beads following the manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA, USA). After this basophil enrichment, primary BM basophils were selected for CD200R3 expression and sorted using a BD FACSMelody™ cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). RNA extraction was performed by using Trizol reagent as described in the manufacturer’s protocol (Invitrogen). cDNA was synthesized with SuperScript™ III First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed with SsoAdvanced SYBR green reaction mix (Bio-Rad, Hercules, CA, USA) using the M_B2m_1 KiCqStart™ primer pair for mouse β2-microglobulin as housekeeping gene (Sigma-Aldrich, Merck) and the following primers for Mcpt8 quantification: Forward primer: 5′-GTGGGAAATCCCAGTGAGAA-3′ and Reverse primer: 5′-TCCGAATCCAAGGCATAAAG-3′ (12 (link)). Quantitative PCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and, following amplification, Ct values were obtained using the CFX Manager™ software 2.1 (Bio-Rad, Hercules, CA, USA).

Real-time PCR was carried out on a CFX96 Cycler real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), in white-walled PCR plates (96 wells). A ready to use master-mix containing a fast proof-reading polymerase, dNTPs, stabilizers, MgCl2 and SYBR^® Green dye was used according to the manufacturer’s instructions (Bio-Rad). Reactions were prepared in a total volume of 18 μL containing 400 nM each primer (MWG), 2 × SsoAdvanced™ SYBR^® Green Supermix (Bio-Rad) and 2 μL cDNA. The cycle conditions were set as follows: initial template denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and combined primer annealing/elongation at 57 °C for 20 s. The amount of fluorescence for each sample, given by the incorporation of SYBR^® Green into dsDNA, was measured at the end of each cycle and analyzed via CFX Manager™ software 2.1 (Bio-Rad Laboratories, Inc.). The gene copies were represented by the expression level of RFP gene divided by that of ALG9 gene which performed as the internal reference gene [40 (link)].

Total RNA was isolated from 5 × 10⁵-1 × 10⁶ cells using the mirVANA™ miRNA Isolation Kit (Life Technologies) according to the manufacturer’s protocol. Primers for ErbB3, RAP1A and GAPDH were QuantiTect Primer Assays from Qiagen, primers for SOS1 (forward: 5’-CGAGCCCTTTTCACTCAAGC; reverse: 5’-GCCATGGGGCAGAGTAACTT). were designed using PRIMER3 [47 (link),48 (link)] and synthesized by biomers.net GmbH, Ulm, Germany. ErbB3, SOS1, RAP1A and GAPDH mRNA levels were quantified by qRT-PCR using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Foster City, CA, USA) according to manufacturer’s protocol. qRT-PCR was performed with a Cfx96 device (Biorad, Hercules, CA, USA). Changes in the relative expression level were calculated using the 2^-ΔΔCt method (Biorad CFX manager software 2.1.). GAPDH was used as the endogenous control gene.