GSM5328921: Control_03; Pinus pinaster; OTHER
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). Nanopore libraries for DRS were prepared from 1.65 up to 2.18 µg of isolated polyA-RNA using the nanopore Direct RNA Sequencing kit (SQK-RNA001, Oxford Nanopore Technologies, ONT, Oxford, UK) according to manufacturer's instructions. The poly(T) adapter ligation to the polyA-RNA was performed using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) in the Quick Ligase reaction buffer (New England Biolabs) for 15 min at room temperature. First-strand cDNA synthesis was carried with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) using the oligo(dT) adapter. The RNA-cDNA hybrid obtained was purified using Agencourt RNAClean XP magnetic beads (Beckman Coulter, Brea, CA, USA). The DRS adapter was ligated to polyA-RNA using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA) in the Quick Ligase reaction buffer (New England Biolabs, Ipswich, MA, USA) for 15 min at room temperature followed by a second purification step using Agencourt beads (as described above). Finally, DRS libraries were loaded onto a R9.4 SpotON Flow Cells (Oxford Nanopore Technologies, Oxford, UK) and sequenced until complete nanopores depletion, around 24 hours of run time.