Casava 1.8.2 version

Total RNA was extracted from Fragaria-derived EPDENs and Fragaria juice using the mirVana™ miRNA Isolation Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Plant RNA Isolation Aid (Thermo Fisher Scientific) was used to remove common plant contaminants such as polyphenols and polysaccharides. RNA-seq analysis was performed by IGA Technology Services (Udine, Italy). The TruSeq Small RNA Sample Prep kit (Illumina, San Diego, CA, USA) was used for library preparation, following the manufacturer’s instructions. Both RNA samples and final libraries were quantified using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quality tested by the Agilent 2100 Bioanalyzer RNA Nano assay (Agilent Technologies, Santa Clara, CA, USA). Libraries were then processed with Illumina cBot for cluster generation on the flow cell, following the manufacturer’s instructions, and sequenced on single-end mode at the multiplexing level requested on HiSeq2500 (Illumina, San Diego, CA, USA). The CASAVA 1.8.2 version (Illumina) of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing. To identify specific miRNA, the miRBase database was used (www.mirbase.org).

DNA recovered from the ChIP procedure was quantified using the Qubit 2.0 Fluorometer (Invitrogen) and the quality was tested by the Agilent 2100 Bioanalyzer (Agilent Technologies). The DNA was then processed, including end repair, adaptor ligation, and size selection, using an Ovation® Ultralow System V2 1–16 (Nugen) sample prep kit following the manufacturer’s instructions. Final DNA libraries were validated and processed with Illumina cBot for cluster generation on the flowcell, following the manufacturer’s instructions and sequenced on single-end 50 bp mode at the on HiSeq2500 (Illumina) at a depth of approximately 30–50 million sequences per sample. The CASAVA 1.8.2 version of the Illumina pipeline was used to processed raw data for both format conversion and de-multiplexing.