GSM5848563: Root at time 24 control experiment 2; Pinus pinaster; miRNA-Seq
Total RNA was isolated using Norgen Biotek Plant/Fungi Total RNA Purification kit (Norgen Biotek, Thorold, ON, Canada). Total RNA concentration and purity were determined with a Nanodrop ND1000 (Thermo Scientific, Waltham, MA, USA). Purity was determined through the 260/280 and 260/230 ratios. The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA BR, Broad-Range, Assay Kit (Invitrogen, Paisley, UK). RNA quality was further confirmed using agarose gel electrophoresis and using the RNA Pico Assay for the 2100 Bioanalyzer (Agilent, USA). RNA samples with an RNA integrity number (RIN) higher than 7 were used for miRNA-Seq. The miRNA isolation and sequencing were carried out by Fasteris (Plan-les-Ouates, Switzerland). Three micrograms of total RNA were used to isolated miRNAs by poliacrilamide gels in order to select sequences with a size between 18 and 30 bp. RNA libraries for sequencing were prepared using the TruSeq small RNA kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The 21 samples were sequenced on an Illumina HiSeq 4000 as single-end reads of 50 bp length.