title: GSM2061053: 2014.SB.MM.N100.1; Vitis vinifera; RNA-Seq
library_construction_protocol: Total RNAs were extracted according to the procedure of Reid et al. (Reid, Olsson, Schlosser, Peng, & Lund, BMC Plant Biol. 2006; 6:27) from 1 g of grounded frozen berries. Traces of genomic DNA were removed by a DNase I treatment (Ambion TURBO DNA-free DNase, Life Technologies). Libraries of template molecules suitable for high-throughput DNA sequencing were created using "TruSeq™ RNA Sample Preparation v2 Kit" (Illumina Inc.). Briefly, mRNA was purified using poly-T oligo-attached magnetic beads and fragmented using divalent cations at 94°C for 8 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers, then the second strand of the cDNA was synthesized using DNA Polymerase I and RNase H. The double-stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double-stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent). The libraries were loaded in the flow cell at 7pM concentration and clusters were generated in the Cbot.
identifiers.namespace: GEO
identifiers.id: GSM2061053