title: RNA-Seq of Pisum sativum: Parafield_Stipule-HiSeq
design: "Parafield plants (conventional plant morphology, large dun-type grain) were maintained in glasshouses at 22 +/- 2C under a 16/8-h (light/dark) photoperiod in individual pots filled with standard potting mix in the premises of DEPI- Bundoora, Victoria, Australia. Stipules (from 4 weeks old), were collected from three plant replicates and after harvest, tissues were frozen immediately in liquid nitrogen and stored at -80C. For RNA extraction (RNeasy Plant Mini Kit [QIAGEN, Hilden, Germany]), replicates from individual tissues were pooled in equal proportion (by weight). The concentration of RNA was confirmed using a spectrophotometer (Thermo-Scientific, Delaware, USA) and the integrity of total RNA was determined by electrophoretic separation on 1.2% (w/v) denaturing agarose gels. For library preparation, polyA-containing (mRNA) fraction was isolated from total RNA (1 ug) using Dynabeads with oligo (dT)25 residues covalently coupled to the surface (Life Technologies Australia Pty Ltd, Victoria, Australia). After purification, mRNA was fragmented by random shearing using heat treatment in the presence of Mg2+ ions and were used as templates to synthesise first-strand cDNA using random hexamer priming and the Bioline Tetro cDNA Synthesis Kit (Bioline US Inc., Massachusetts, USA). Second strand synthesis was performed in a solution containing NEBuffer 2 (New England Biolabs Ltd., Hitchin, United Kingdom), dNTPs (Bioline US Inc.), RNaseH (New England Biolabs) and DNA polymerase I (Thermo-Scientific). Subsequently, the ends of nascent cDNAs were polished by the addition of T4 DNA Polymerase (New England Biolabs) and Klenow DNA Polymerase (New England Biolabs) followed by paired-end adapter ligation to both termini of the cDNA fragments. Templates were then subjected to amplification using Phusion (Thermo-Scientific) DNA polymerase. The amplified libraries were pooled in equimolar amount and assessed by loading of 1 ul aliquot on an Agilent Bioanalyzer 1000 DNA chip according to the manufacturer's instructions. Library quantification was performed using the KAPA library quantification kit (KAPA Biosystems, Boston, USA). All reads were pair-end sequenced using the HiSeq 2000 and MiSeq platforms (Illumina Inc., San Diego, USA)."