GSM5825890: Somatic embryos cotyledonary stage 3 sample  C; Pinus pinaster; RNA-Seq
Total RNA of embryo samples 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. RNA-seq was carried out by Novogen (UK). mRNA was enriched using oligo(dT) beads. First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. RNA sequencing was made in a NovaSeq 6000 sequencer according to the manufacturer's instructions for paired-end reads (Illumina, San Diego, CA, USA). The samples were sequenced producing paired-end reads of 150 bp length.