title: GSM8148779: Grape berry. Cabernet sauvignon genotype, Sicily site rep1; Vitis vinifera; RNA-Seq
library_construction_protocol: Ten berries from each biological replicate, kept frozen by continuously liquid nitrogen adding, were ground using a precooled mortar and pestle. Total RNA was isolated from ground whole berries, excluding seeds, by using a slight modification of the method described by Japelaghi and co-workers (Japelaghi et al., 2011). Briefly, a scale-up of the ground tissue was needed: in detail 1 g of sample was added to 10 ml of extraction buffer. Furthermore, three steps of the chloroform: isoamyl alcohol (24:1) extraction were performed. Sequen_x0002_cing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ips_x0002_wich, MA, USA) following manufacturer's recommenda_x0002_tions. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using ran_x0002_dom hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subse_x0002_quently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, MA, USA). Then 3 μl USER Enzyme by NEB was used with size-se_x0002_lected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.