Lightcycler 480 2

10 to 20 ng of DNA of each patient were used for TaqMan SNP genotyping assays (comprising primers and fluorescent probes) according to the manufacturer’s instructions (Thermofisher Scientific, Waltham, MA USA). Genotyping was performed by real–time PCR using either allelic discrimination in the 7300 RT–PCR System (Thermofisher Scientific, Waltham, MA USA), either using endpoint genotyping in an LightCycler 480II (Roche Molecular Diagnostics Pleasanton, CA, USA). For the 7300 RT–PCR System PCR parameters involved an initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Post run data were analysed by 7300 SDS software (Thermofisher Scientific, Waltham, MA USA) and Automatic calls were assigned with approximately 99.8% quality. A call rate > 95% was considered the cutoff to consider genotyping. For LightCycler 480II PCR parameters involved an initial denaturation at 95 °C for 10 min followed by 40 cycles at 95 °C for 10 s, 60 °C for 1 min, and 72 °C for 10 s. Post run data were analysed by LightCycler 480 Endpoint Genotyping software (Roche Molecular Diagnostics Pleasanton, CA, USA). Two blank (water) controls in each 96-well plate were used for the assay quality control.

The total RNA was isolated using NucleoSpin kit reagents (Macherey-Nagel GmbH & Co. KG, Hoerdt, France), according to the manufacturer’s protocols. The RNA was quantified by spectrophotometry (Nanodrop, Thermo Fisher, Illkirch, France), and cDNA was prepared using 2 µg of total RNA, random hexamers, and SuperScript IV reverse transcriptase (Thermo Fisher, Illkirch, France), following the manufacturer’s instructions. Quantitative PCR (Light Cycler 480-II) was performed using the Light Cycler 480 SYBR Green I Master X2 Kit (Roche Diagnostics, Meylan, France), according to the supplier’s protocol. The data were analyzed using the standard curve method, following the manufacturer’s protocol (Lightcycler 480 II, Roche Diagnostics, Meylan, France), and the 18S housekeeping gene as internal control.

A total of 1 μg of RNA was used for cDNA synthesis using SuperScript III following the manufacturer’s protocol (Invitrogen, Gaithersburg, MD, United States). Then, the FPKM values for nine randomly selected genes were validated by reverse transcription quantitative real-time PCR (RT-qPCR) using the LightCycler 480II (Roche, Mannheim, Germany). A total of 45 ng of cDNA was used as a template for RT-qPCR with gene-specific primers (Supplementary Table 1) using 2 × SyGreen Mix (qPCRBIO Lo-ROX) (PCR Biosystems, London, United Kingdom). Thermocycling conditions were 95°C for 5 min, 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. At the end of the PCR cycles, the Ct values were analyzed using LightCycler 480II software (Roche). The efficiency of each gene-specific primer was determined using pooled cDNA samples. The expression of each gene was normalized using the comparative 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)) with BrActin as a reference gene.

RNA‐Seq data were verified by qRT‐PCR using specific primers of selected genes (Table S7). Bacterial growth, RNA isolation and cDNA synthesis were as described above for RT‐PCR and RNA‐Seq experiments. qRT‐PCRs were performed in a Light Cycler 480 II (Roche, Basel, Switzerland) using 1 μg cDNA, 0.6 pmol of each primer and the HOT FIREPol EvaGreen qPCR Mix Plus (Solis BioDyne, Tartu, Estonia), and the following conditions: 95 °C for 15 min (1 cycle); 95 °C for 15 s, 60 °C for 20 s and 72 °C for 20 s (40 cycles); melting curve profile from 65 to 97 °C to verify the specificity of the reaction. The A. citrulli GAPDH gene was used as an internal control to normalize gene expression. The threshold cycles (C_t) were determined with the Light Cycler 480 II software (Roche) and the fold‐changes of three biological samples with three technical replicates per treatment were obtained by the ΔΔC_t method (Pfaffl, 2001). Significant differences in expression values were evaluated using the Mann–Whitney nonparametric test (α = 5%).

Total RNA was extracted using RNA-IsoPlus (Takara) and cDNA was synthesized by PrimeScript RT Master Mix (Takara). qPCR was performed with EvaGreen qPCR master mix (ABM) and primers listed in Supplementary Table 2 on a LightCycler 480 II analyser (Roche) with data analysed using the LightCycler 480 II software (Roche). Relative expression differences were calculated using the 2^-ΔΔCt method.

All samples were genotyped for MDM4 SNP34091 status using a custom LightSNiP assay (TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) on a LightCycler 480 II instrument (Roche, Basel, Switzerland). The reactions were performed in a final reaction volume of 10 μL, containing 1 μL LightCycler^®FastStart DNA Master HybProbe mix (Roche Diagnostics), 0.5 μL LightSNiP mix (TIB MOLBIOL), 3 mmol/L MgCl₂ and 10–50 ng DNA. The thermocycling and melting curve conditions were as follows: 10 min initial denaturation/activation at 95°C, followed by 45 cycles of denaturation at 95°C for 10 sec, annealing for 10 sec at 60°C and elongation at 72°C for 15 sec. Subsequent to the thermocycling amplification the high‐resolution melting (HRM) step was initiated with a denaturation step at 95°C for 30 sec, followed by melting from 40°C to 75°C with a ramp rate of 0.19°C/sec and finally a cooling step at 40°C for 30 sec. The HRM curve profiles were analyzed by the Melt Curve Genotyping software (version 1.5) on the LightCycler^® 480 II instrument (Roche Diagnostics).