GSM5993420: Needles 8mM nitrate at 400 ppm of CO2 replicate 1; Pinus pinaster; 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). RNA sequencing was conducted by aScidea Computational Biology Solutions S.L. sequencing company (Barcelona, Spain). Only RNA samples with a RIN > 7 in a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) were used for library preparation. RNA libraries for sequencing were prepared starting with 1 µg of total RNA and using TruSeq RNA kits (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Clustering was performed on a cBot cluster generation system using an Illumina NovaSeq paired-end read cluster generation kit according to the manufacturer's instructions (Illumina, San Diego, CA, USA). The samples were sequenced on an Illumina NovaSeq 6000 as paired-end reads of 150 bp length.