Dsdnase kit

The cDNA was prepared by reverse transcription of 1 μg of total RNA using the Maxima cDNA Kit with the dsDNase kit (Thermo Fisher Scientific, Waltham, MA, United States) following the manufacturer’s instructions. Quantification was performed by real-time quantitative RT-PCR (qPCR) using iCycler iQ^™ Real-Time PCR Detection System (BIORAD) and quantified using Real-Time Detection System Software (version 2.0). The amplification reactions were carried out in a final volume of 12 μl containing 6 μl of NZY qPCR Green Master Mix (2×; NZYTech, Ltd), 1 μl of each primer (10 μM), and 1 μl of the cDNA. The PCR profile used was 2 min at 50°C, 95°C for 10 min, followed by 40 cycles of 20 s at 95°C and 30 s at 55 or 60°C. Three technical replicates were made from each of the genes studied. Gene expression was determined by the 2^−ΔΔCT method using the F. vesca Actin-97-like (XM_004307470; FvH4_7g22410; gene26612) as a housekeeping gene. The Fragaria vesca eFP Browser (Darwish et al., 2013 (link)) provided us with a basis from which we selected FvACT as a housekeeping gene (Hollender et al., 2014 (link)), since it shows less variability in expression for all developmental stages studied than other housekeeping genes. The specific primers used are described in Supplementary Table S1 and PCR amplicons were sequenced to confirm specificity.

After infection with PA for 24 h, total RNA was isolated using a GeneJET RNA Purification Kit (Thermo Scientific, Waltham, MA, USA, K0732). RNA concentrations in each sample were assessed using a Nanodrop (Thermo Scientific) and cDNA was synthesized using a Maxima^TM H Minus cDNA Synthesis Master Mix with a dsDNase kit (Thermo Fischer, Waltham, MA, USA, K1652). RT-qPCR was performed on an Applied Biosystems StepOnePlus using the TaqMan primers Interleukin-8 (Hs00174103_m1, CXCL8), Interleukin-6 (Hs00174131_m1, IL-6), Interleukin 1-β (Hs01555410_m1, IL-1β), FGFR1 (Hs00241111_m1, FGFR1), FGFR2 (Hs01552918_m1, FGFR2), FGFR3 (Hs00179829_m1, FGFR3), FGFR4 (Hs01106910_g1, FGFR4), Transforming growth factor-beta (Hs00998133_m1, TGFB1), klotho (Hs00934627_m1, KL), and reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Fold changes were calculated via the ΔΔCT method previously described [23 (link)].