Illumina RNA-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. cDNA libraries for Illumina RNA-seq were prepared separately for each replicate. mRNA was purified with oligo(dT) magnetic beads, fragmented, and reverse-transcribed with random hexamer primers, followed by second-strand synthesis. Libraries were prepared through end repair, A-tailing, adaptor ligation, size selection, amplification, and purification.