UVB exposure after 3 hours recovery (replicate 1)
Organic peach fruits (Prunus persica L., cv Fairtime), uniform in size (8.1 cm average diameter) and colour, and without any wound or damage, were purchased from a local organic supermarket.The UV-B treatment was conducted at room temperature (24 C) using UV-B tubes together with white light tubes.Control fruits were placed in a separated climate chamber at the same temperature conditions but exposed to just white light.The UV-B exposure lasted 60 min, corresponding to a total irradiance of 38.53 kJ m2 (8.33 kJ m2 UV-B + 30.20 kJ m2 white light), while control fruits were given a total irradiance of 30.20 kJ m2 white light.Randomized groups of five peaches from both the control and the UV-B-treated groups were sampled after one and three hours from the end of the UV-B treatment.During the recovery period, the fruits were kept at the same climate chambers at room temperature (24 C) with just the white light on.Then, the peach peel was accurately removed with scalpels and tweezers, immediately dipped into liquid nitrogen, and kept at -80 C.RNA was extracted from lyophilized samples with the LiCl/CTAB method .Briefly, a pre-heated extraction buffer (2% [w/v] hexadecyltrimethylammonium bromide, CTAB; 2% [w/v] polyvinylpyrrolidone (average molecular weight 40000), PVP; 100 mM Tris/HCl pH 8.0; 25 mM EDTA; 2 M NaCl; 0.5 g/L spermidine and 2.7% [v/v] 2-mercaptoethanol) was added to the freeze-dried material,and ice-cold chloroform:isoamyl alcohol (24:1) was used for phase separation. Addition of 10 M LiCl at 4 C with following overnight incubation allowed RNA selective precipitation. After rinsing with cold 70% EtOH and re-hydration in 100 L RNAse free water, the RNA samples were stored at -80 C. RNA concentration and integrity were checked through Qubit RNA HS Assay Kit (Invitrogen) and WPA Biowave spectrophotometer (Biochrom Ltd.,Cambridge, England), and through agarose gel electrophoresis, respectively. Enzymatic digestion to get rid of contaminant genomic DNA was performed using RQ1 DNAse (Promega) according to the manufacturers protocol.RNA-Samples were sequenced with Illumina HiSeq 2500.