GSM5962391: First biological replicate of the hybrid G x Q; Solanum lycopersicum; RNA-Seq
Total RNA was extracted from three red ripe fruits (biological replicates) per genotype with Tripure Isolation Reagent, following the manufacturer's instructions (Roche, Basel, Switzerland) with some minor modifications. It is worthwhile to mention that the samples used for RNA-seq and 1H-NMR were obtained from the same biological material, an important aspect to consider for omics data integration (Cavill et al., 2016). For each sample, 100 mg of frozen mesocarp powder was weighted in a sterile 1.5 ml polypropylene centrifuge tube. Then, 950 µl of cold Tripure Isolation Reagent was added and the sample was homogenised and centrifuged for 7 min, 4 to 10 °C, 12,000 × g. The supernatant obtained was transferred to a sterile 1.5 ml tube with 200 µl of chloroform at room temperature to phase separation. The sample was shaken vigorously for 15 sec and incubated for 5 to 15 min at room temperature. Tubes were centrifuged for 10 min, 4 to 10 °C, 12,000 × g. The colourless upper aqueous phase was transferred to a new sterile 1.5 ml tube. To precipitate RNA, 500 µl of cold isopropanol was added to each tube and it was inverted several times to mix it thoroughly. Samples were incubated for 20 min on ice. Tubes were centrifuged for 10 min, 4 to 10 °C, 12,000 × g. The supernatant was discarded. To wash the RNA pellet, 200 µl of 80% ethanol was added to each centrifuge tube. The samples were vortexed and centrifuged for 5 min, 4 °C, 7,500 × g. Excess of ethanol was removed from the RNA pellet by air-drying for 5 to 10 min. The RNA pellet was resuspended in 40 µl of diethylpyrocarbonate (DEPC)-treated RNase-free water. Finally, the samples were vortexed and centrifuged for 1 min, 4 °C, 7,500 × g. The quality and concentration of total RNA extracted were quantified using a NanoDrop Lite Spectrophotometer (Thermo ScientificTM, USA). The RNA integrity was checked using 1.5% denaturing formaldehyde agarose gel-electrophoresis with 1X MEN buffer (0.02 M MOPS + 5 mM NaOAc + 1 mM EDTA) stained with ethidium bromide. Each sample consisted of 5 µl RNA solution (2 µg RNA with ultrapure sterile water) with loading buffer (5 µl formamide, 1.25 µl 10 X MEN, and 1.5 µl formaldehyde), and was heated 10 min at 65 °C. After quality and quantity assessment, 3 µg of RNA in each sample was diluted with DEPC-water to reach a final volume of 30 µl. Then, 0.1 volume of 3 M Sodium Acetate (NaOAc) pH 5.5 and 2 volumes of 100% ethanol were added to the 30 µl of RNA solution for precipitation, mixing gently after each step. The cDNA library construction with fragments of 250-300 bp and the paired-end-sequencing (2 x 150 bp) services were hired from Novogene Corporation, Inc. (Sacramento, CA, USA), using an Illumina Novaseq 6000 platform.