PacBio Iso-seq of Lupinus angustifolius L. leaf and stem transcriptomes
Fully expanded young leaves from plants that had reached the whole plant flowering stage were collected into sterile plastic tubes, immediately flash frozen in liquid nitrogen and stored at −80 °C until nucleic acid extraction. Immediately after leaf sampling, stem tissue was collected from the same plants by excising segments from the upper, middle and lower parts of the main stem. Stem segments were placed into sterile microcentrifuge tubes, flash frozen in liquid nitrogen and stored at −80 °C until RNA isolation. Plants were cultivated in a growth chamber (16 h photoperiod, 22/18 °C day/night, 55–60% relative humidity, until they reached whole plant flowering stage. Total RNA was isolated from 30 mg of frozen, ground organ tissue using a Maxwell RSC Plant RNA Kit (Promega, Madison, WI, USA). RNA concentration and integrity were assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), with a minimum RNA integrity number of 8. Total RNA from five plants per accession was pooled in equal amounts to prepare a single Iso-Seq library. Libraries were constructed following PacBio's Iso-Seq sequencing protocol combining non-size selected and size selected fractions. The first strand cDNA was synthesized using the SMARTer PCR cDNA Synthesis Kit (Clontech, CA, USA), amplified, and processed with the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, CA, USA).