title: RNA-seq reads from the grass species Koeleria macrantha, Jan 2014
library_construction_protocol: Four RNA-seq libraries were created, one for each of four species, Carex panicea, Festuca ovina, Koeleria macrantha and Lotus corniculatus). For each species, RNA was isolated from floral tissue (early and late in ontogeny), leaf tissue (early and late in ontogeny), stem tissue, root tissue, and rhizome tissue (the latter, for Carex panicea and Lotus corniculatus only). RNA was extracted using TRIzolTM reagent, or the Purelink RNA Mini Kit (Ambion), or using CTAB extraction buffer with organic solvent clean up and lithium chloride precipitation (Gambino et al., 2008; Phytochemical Analysis, 19, 520ï¿½525), depending on tissue type and species. Extracted RNA from different tissues was pooled within species to create a single sample (four samples in total). RNA quality, quantity and integrity were checked via analysis on the nanodrop platform (ThermoFisher Scientific), via qubit assays (ThermoFisher Scientific), and on the Agilent Fragment Analyzer platform. RNA samples were treated with DNaseI and cleaned up using the Purelink RNA mini kit. RNA yield, quality and integrity were checked again, as above. Each of the samples was rRNA depleted, using the Ribo-Zero Magnetic Kit for Plant Leaf (Cat. No. MRZPL116; Epicentre, Illumina). The efficacy of rRNA depletion was evaluated using the Agilent Fragment Analyzer platform, comparing samples pre- and post-depletion. The quantity and quality of the resulting RNA samples was also checked by nanodrop fluorimetry and on the qubit platform. The four samples were used to make stranded (directional) sequencing libraries, using the ScriptSeq mRNA-Seq Library Prep kit (Epicentre), with a unique index for each library. The four libraries were pooled and sequenced on one lane of the Illumina HiSeq1000 platform, using 2 x 100 bp paired-end reads.