title: De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes
design: Plant materials and library construction: The seed of three pepper (C. annuum) lines ‘CM334,’ ‘Maor’ and ‘Early Jalapeño’ (EJ) were planted in the greenhouses of the Department of Plant Sciences at UC Davis under standard conditions for Capsicum [27] until adult stage. Three cDNA libraries (one from each pepper variety) were prepared using pooled mRNA that was independently extracted from seven tissues: root, young leaf, 5, 10, and 20 days post pollination developing fruit, breaker and ripe fruit using Qiagen RNeasy Mini Kit (Qiagen Valencia CA, USA) per the manufacture’s protocol. CM334 root tissue was inoculated with Phytophthora capsici to induce expression of resistance genes. Aliquots were quantified using a NanoDrop spectrophotometer (NanoDrop Wilmington, USA) and checked for quality by electrophoreses separation using Lonza FlashGel System FlashGel RNA Cassettes (Lonza Inc. Allendale,,USA). Samples were pooled in equivalent concentration. For each pepper line paired-end libraries were prepared following standard Illumina protocols (Paired-End DNA Sample Prep Kit). The libraries were sheared and 300-350 bp fragments were selected on gels. The libraries were normalized using double-stranded nuclease to digest high copy double-stranded DNA during re-association after denaturation and then prepared for sequencing as described in Illumina [28]. The cDNA libraries were sequenced using Illumina Genome Analyzer IIx (IGA) for 85 cycles per direction at UC Davis Genome Center. One lane of paired-end pass and one lane of single pass were run for each of CM334 and Maor lines and two lanes of paired-end pass were run for Early Jalapeño.