title: Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea
design: Plants were grown in a growth chamber (photoperiod 16h light/day, 15°C night, 20°C day, hygrometry 60% min) and whole plants were collected at 15 days after sowing. At least 5 plants were pooled. Tissues were flash frozen in liquid nitrogen and stored at -80° until further experiment.  Total RNA was extracted from tissue powder with RNeasy plant kit (Qiagen) according to manufacturer’s instructions. RNA purity and integrity were checked by capillary electrophoresis on a BioAnalyzer (Agilent). RNA concentration was determined by spectrometry on a Nanodrop instrument (fournisseur), and OD260/OD280 ratio calculated for purity assessment. cDNA normalization was performed from total RNAs with MINT and TRIMMER kits from Evrogen according to manufacturer’s instruction, except that we adapted to our material the number of PCR cycles for material amplification. First, full length double stranded (ds) cDNA were synthetized from 2µg of total RNA using MINT kit (Schmidt et al., 1999). First strand was synthetized from a fusion primer containing an oligo(dT) stretch to anneal to RNA polyA tails. A poly(dC) stretch was incorporated at the end of the first strand, and used for priming the synthesis of the second strand. Full length (ds) cDNA were subsequently amplified by PCR, purified on Qiaquick columns (Qiagen) and checked for quality and yield before normalization.  Normalization was done with TRIMMER kit (Evrogen) which is based on DSN technology (Zhulidov et al., 2004). The method involves denaturation-reassociation of cDNA, Duplex Specific Nuclease (DSN) degradation of ds-fraction corresponding to abundant transcripts and PCR amplification of single strand (ss) DNA fraction. We started from 600ng (ds) cDNA for normalization and after denaturation, incubated samples at 68°C for 5 hours for renaturation. After degradation of (ds) complexes by DSN, we made 2 runs of PCR amplification for optimal recovery. Normalized cDNA was then purified on Qiaquick columns (Qiagen) and yield was measured by spectrophotometry. Sequencing library preparation was performed using Roche 454 GS-FLX kits according to manufacturer’s recommendations. We started with 1µg (ds) cDNA that was submitted to fragmentation using nebulization method (Roche). An average size of 700pb was obtained for each sample, as verified by capillar electrophoresis (Agilent Bioanalyzer). Library was sequenced on a 454 GS-FLX sequencer (Roche) with the Titanium chemistry (400bp read length) on half a PicoTiterPlate (PTP).