RNA-Seq of Phillyrea angustifolia: pollinated pistils
The TruSeq RNA sample Preparation v2 kit (Illumina Inc., USA) was used according to the manufacturer's protocol with the following modifications. In brief, poly-A containing mRNA molecules were purified from 1 ug total RNA using poly-T oligo attached magnetic beads. The purified mRNA was fragmented by addition of the fragmentation buffer and was heated at 94C in a thermocycler for four minutes to yield library fragments of 250-500 bp. First-strand cDNA was synthesized using random primers to eliminate the general bias towards 3'end of the transcripts. Second strand cDNA synthesis, end repair, A-tailing, and adapter ligation was done in accordance with the manufacturers protocols. Purified cDNA templates were enriched by 15 cycles of PCR for 10s at 98C, 30s at 65C and 30s at 72C using PE1.0 and PE2.0 primers and with Phusion DNA polymerase (NEB, USA). Final pooled cDNA libraries were sequenced using the Illumina mRNA-Seq, paired-end protocol on a HiSeq2000 sequencer, for 2 x 100 cycles.