title: Voodoo Lily 454 deep sequencing
design: Frozen corm material was ground to a fine powder using mortar and pestle. Total RNA was extracted from 4.18g of ground tissue using the pine tree method published by Chang et al. 1993. DNA was digested and removed using Qiagen RNase free DNase I (catalog #29254, Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The RNA quality was checked and assured using Agilent Bioanalyzer RNA pico 6000 chip (Agilent, Santa Clara, USA).  First strand cDNA was synthesized using the ClonTech SMART cDNA library construction kit (catalog #63490, ClonTech, Mountain View, CA, USA) according to the manufacturer’s instructions. A modified version of the CDS III/3’ primer was used (5’- TAG AGG CCG AGG CGG CCG ACA TGT TTT GTT TTT TTT TCT TTT TTT TTT VN-3’, Integrated DNA Technologies, Coralville, IA, USA). Reverse transcription was realized using Invitrogen SuperScript II Reverse Transcriptase (catalog #18064-022, Invitrogen, Carlsbad, CA, USA).  The cDNA was amplified by ligation-dependent PCR (LD-PCR) using the ClonTech Advantage 2 Polymerase mix (catalog #639201, ClonTech) according to the manufactures recommendations. The amplified cDNA was digested with SfiI, size fractionated and precipitated using ClonTech SMART cDNA library construction kit (catalog #63490, ClonTech) according to the manufacturer’s instructions.  A total of 10µg cDNA was submitted for Roche® 454 GSFLX Sequencing. The 454 sequencing was carried out by Michigan State Universities Research Technology Support Facility Genomics Core (Michigan State University, East Lansing, MI, USA).