GSM6336769: Thinning and Shading, May 2017, rep 2; Abies pinsapo; RNA-Seq
Total root RNA from maritime pine seedlings was isolated following the protocol described by Liao et al. (2004, Preparative Biochemistry & Biotechnology 34: 209–214) and modified by Canales et al. (2012, Frontiers in Plant Science 3: 100). The RNA concentration and purity were determined via spectrophotometry on a Nanodrop ND-1000 (Thermo Scientific, Waltham, MA, USA). Purity was determined through the 260/280 and 260/230 ratios. RNA quality was also determined in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA BR, Broad-Range, Assay Kit (Invitrogen, Paisley, UK). Samples with a RIN value > 7 were selected to mRNA isolation. The polyA-RNA isolation was performed using Dynabeads™ mRNA Purification Kit (Invitrogen, Paisley, UK) following the manufacturer's instructions. This process was carried out twice per sample to avoid rRNA contamination. polyA-RNA quality was determined in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The concentration was verified with a Qubit 4 Fluorometer (Invitrogen, Paisley, UK) and Qubit RNA HS, High Sensitivity, Assay Kit (Invitrogen, Paisley, UK). The sample sequencing was made by means of the company AscideaCro Genomics and Bioinformatics. The mRNA library preparation for sequencing were prepared using TruSeq RNA kits (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The samples were sequenced on an Illumina Hiseq 4000 sequnecer (Illumina, San Diego, CA, USA) according to the manufacturer's instructions obtaining paired-end reads of 150 bp length.