title: GSM8115276: flg22 treated plant 6h bio rep 3; Prunus dulcis; RNA-Seq
library_construction_protocol: Sampling was carried out 6 and 24 hpt and for each replicate 20 leaves were pooled (4 leaves/plant) and they were immediately frozen in liquid nitrogen. The material was finely grounded and homogenized with a Tissue Lyser II. RNA was extracted from 100 mg of the grounded leaf material coming from each replicate using the PureLink™ Plant RNA Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. The remaining DNA was digested with the TURBO DNA-freeTM Kit (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA “TruSeq Stranded mRNA Sample Prep kit” (Illumina, San Diego, CA) was used for library preparation following the manufacturer's instructions, starting with 1-2 µg of good quality RNA (RIN > 7) as input. The RNA was fragmented 3 min at 94°C and every purification step was performed using 0.81X Agencourt AMPure XP beads. Both RNA samples and final libraries were quantified using the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quality tested by Agilent 2100 Bioanalyzer RNA Nano assay (Agilent technologies, Santa Clara, CA, USA). Libraries were then processed with Illumina cBot for cluster generation on the flowcell, following the manufacturer's instructions and sequenced on paired-end (2x150 bp, 30M reads per sample) at the multiplexing level requested on NovaSeq6000 (Illumina, San Diego, CA, USA).