title: ESTs of Chenopodium quinoa from seed, salt-stressed, and non-stressed tissues
design: Quinoa seeds, provided by A. Bonifacio (PROINPA, LA Paz, Bolvia), of the Salares ecotype ‘Ollague’ were germinated in water for 4-5 days at room temperature. The seedlings were then transferred to hydroponic growth chambers using Hoaglands solution as described by Camp et al. [18] with day temperature of 29.5°C, night temperature of 19°C, and day length of 13 hours. In order to obtain salt stress transcriptomes, NaCl was added to the Hoaglands growing solutions for the following treatments: 1) control - 0 mM NaCl; 2) low salt stress - 150 mM NaCl; 3) intermediate salt stress - 300 mM Nacl; and 4) a high salt stress - 450 mM NaCl. Plants in the stress treatments were supplemented with NaCl at 50mM increments on a daily basis (starting 14 days after germination), until the hydroponic solution reached the final concentration for each treatment. After the final salt concentration was attained, the plants were grown for 4 days until tissue harvest.    Root, leaf and flower tissue were harvested simultaneously, prior to anthesis. Tissues were flash frozen in liquid nitrogen and RNA was extracted using the hot borate method [19] from approximately 0.1 g of each tissue type. The extracted RNA was analyzed for quality and quantity using an Agilent 2100 Bioanalyzer and RNA NanoChip with 2100 Expert software. Total RNA from each tissue type and treatment were pooled together in equimolar amounts (400 ng each). The pooled RNA sample was treated twice with the Ribominus™ Plant Kit for RNA-Seq (Invitrogen, Carlsbad, CA) and a pooled cDNA library was constructed according to the cDNA Rapid Library Preparation Method Manual and sequenced using standard protocols for 454-pyrosequencing as a service at the Brigham Young University DNA Sequencing Center (Provo, UT) using a Roche-454 GS FLX instrument and Titanium reagents and GS FLX Titanium General Library Preparation Kit (Roche, Branford, CT).