GSM6599907: P2_t24 rep1; Prunus persica; miRNA-Seq
RNA was extracted from a 200 mg aliquot of ground leaf sample through a TRIzol-based procedure (Invitrogen Life Technologies, Carlsbad, CA, USA) followed by DNAse I (Ambion, Grand Island, NY, USA) digestion and purification with RNeasy MinElute Cleanup Kit (Qiagen, Sollentuna, Sweden). Estimation of RNA concentration was using a NanoDrop ND1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). miRNA-Seq was carried out at the National Centre for Genomics Analysis (Barcelona, Spain). Libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina® kit (ref. E7330) according to the manufacturer's protocol. Briefly, RNA was subjected to adaptor 3´and 5´ ligation and first strand cDNA synthesis. Then, DNA fragments having adapter molecules on both ends were selectively enriched by PCR. Library amplification was performed by using NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1, ref. E7335, Index Primers Set 2, ref. E7500, Index Primers Set 3, ref. E7710 and Index Primers Set 4, ref. E7730). Purification steps were then performed using AgenCourt AMPure XP beads (ref. A63882, Beckman Coulter) and final libraries were analyzed using Agilent Bioanalyzer (ref. 5067-4626) to estimate the quantity and check size distribution. A pool was done to perform size selection using 6% Novex TBE PAGE Gels (ref. EC6265BOX) and then the final pool was quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina's cBot. Libraries were sequenced 1 * 50+8 bp on Illumina HiSeq2500 device