title: Global transcriptome analysis reveals significant differences in gene expression patterns between the non-hyperhydric and hyperhydric leaves of Prunus persica
design: Total RNAs from non-hyperhydric and hyperhydric leaves of peach plantlets were isolated using TRIzol Reagent (Invitrogen, CA, USA) according to the protocol provided by the manufacturer. Residual genomic DNA was eliminated from each RNA samples using DNase I digestion (Thermo Scientific). The concentration and quality was initially assessed using a Nanodrop ND-2000c spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) at an absorbance ratio of A260/230 and A260/280 nm. Then, RNA integrity was evaluated by analysis on the Agilent 2100 bioanalyzer (Agilent Technologies) and only the samples with a RIN (RNA integrity number) > 8.0 were used for subsequent experiments. RNA-Seq library preparation and sequencing reactions were conducted in Genetic Engineering and Biotechnology Institute (GEBI) of TUBITAK according to the “TruSeq RNA Sample Preparation Guide”. Briefly, poly-A mRNAs were purified from 2 μg total RNA using oligo (dT) attached magnetic beads and chemically sheared into short fragments via fragmentation buffer. Sheared fragments were converted to double-stranded cDNA using random hexamer primers and reverse transcriptase (Superscript II, Invitrogen). Second strand cDNA was synthesized using DNase I and RnaseH. Subsequently, end repair and single A-base tailing were performed before Illumina adapters were ligated to the cDNA fragments. After agarose gel size fractionation step to extract fragments of ∼200 bp, cDNA libraries were enriched by PCR amplification. Following this step, the prepared libraries sequenced using the Illumina HiSeq 2000 platform with paired-end 101-bp reads. Base calling was performed using Illumina instrument software.
identifiers.namespace: Istanbul University
identifiers.id: Prunus persica leaf transcriptome