title: Transcriptome analysis of Vicia Sativa spp. nigra to develop useful genomic resources for the sustainable agriculture
design: Vicia sativa subsp. nigra seedlings were cultivated in a glasshouse, and the leaves of the young seedlings were processed to extract mRNA. Total RNA isolation was performed using a Trizol RNA isolation protocol (modified by D. Francis from Edgar Huitema) and the RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s manual. Young seedling leaves (100 mg) were placed in liquid nitrogen, ground into a powder, and subjected to total RNA extraction. Total RNA density was determined using a Biospec-Nano spectrophotometer (Shimadzu, Kyoto, Japan) and agarose gel electrophoresis. mRNAs were purified with the PolyATract mRNA Isolation System (Promega, Madison, WI, USA). The purified products were used to synthesize the full-length cDNA using the ZAP-cDNA Synthesis kit (Stratagene, Santa Clara, CA, USA). The cDNA was fragmented by nebulization using an Agilent 2100 bioanalyzer (Waldbronn, Germany) with a mean fragment size of ~600 bp. Approximately 1-µg cDNA was used to generate a library for genome sequencing with an FLX Titanium analyzer (Roche, Mannheim, Germany). The cDNA fragment ends were blunted, and two short adapters were ligated to each end according to standard procedures (Margulies et al. 2005). The adapters provided priming sequences for amplification and sequencing of the sample library fragments. They also served as a sequencing key, which is a short sequence of four nucleotides used by the system software for base calling. The sequencing key also released the unbound strand of each fragment (with 5-adaptor A) following repair of any nicks in the double-stranded DNA library. The quality of the single-stranded template DNA fragment library was assessed using the 2100 bioanalyzer, and the library was quantitated, including functional quantitation, to determine the optimal amount to use as input for emulsion-based clonal amplification.