RNAseq of fruit
RNA sequencing was performed using the Ion Proton system. Total RNA was enriched for poly-A mRNA fraction by Dynabeads mRNA DIRECT Micro Kit (Thermo Fisher Scientific, Waltham, MA, USA). The mRNAs isolated were used for RNA-seq library preparation using the procedure for low-input RNA in Ion Total RNA-Seq kit v2 (Thermo Fisher Scientific, Waltham, MA, USA). The procedure follows RNA fragmentation using RNase III enzymatic digestion and ligation of Ion Adapters using four different barcodes to retain tissue specificity. Samples were reverse transcribed, purified, cDNA amplified and obtained library checked by the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CO, USA). Libraries were in equimolar amounts pooled together and amplified in emusion PCR by Ion OneTouch 2 System using Ion PI Hi-Q OT2 200 Kit. Template-positive particles were enriched by using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher Scientific, Waltham, MA, USA) on an Ion OneTouch ES system. The obtained enriched particles samples were sequenced on PI Chip v3 using the Ion PI Hi-Q Sequencing 200 Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer protocol. The raw sequencing reads quality and adapter trimmed were obtained.