RNA-Seq of Erythrina velutina: adult leaves
Seeds and leaves from Erythrina velutina were ground to a very fine powder with the aid of a mortar and pestle. 100 mg of each powder sample were used for RNA extraction. The total RNA of seeds and leaves was isolated using the TrizolTM Plus RNA Purification Kit (Life Technologies) following manufacturer instructions. To obtain high quality samples without the presence of genomic DNA and other contaminants, the samples were treated with RNAse Free DNAse (Qiagen) and subsequently purified with the RNeasy Mini kit (Qiagen). RNA quality was verified using a 2100 Bioanalyzer RNA Nanochip (Agilent) and all samples that had an RNA integrity number (RIN) value above 6.0 were selected. To define the quantity needed to compose the sample pools, the selected RNA samples were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop) and Qubit fluorometric quantification (ThermoFischer Scientific). Each pool was composed of three RNA samples and a total of 0.5 g of RNA equally grouped for the preparation of cDNA libraries. The sample sets were indexed with the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's protocol (Illumina). The libraries were quantified using the Kapa Library Quantification Kit for the Illumina Genome Analyzer platform (Kapa Biosystems) on an Applied Biosystems ViiA7 machine (Life Technologies), according to the manufacturer's instructions. The same amount of each RNAseq library was grouped to equalize the index concentration and then submitted for 150 bp paired end sequencing according to the standard protocols for a NextSeq500 platform (Illumina).