Illumina HiSeq 2500 paired end sequencing; RNA-seq of bird-pollianted, red-flowering and insect-pollinated, yellow-flowering Diplacus aurantiacus varieties sourced from various locations in San Deigo State, USA
Diplacus aurantiacus plants were obtained by stem cuttings taken from four naturally occurring populations in San Diego County (California, US). The species is not on the list of endangered/protected plants and no specific permission is required to use this material. M. aurantiacus was grown and maintained in pots in the greenhouse at a day/night temperature of 20/18ºC with a 16-h photoperiod and a relative humidity of 60%. For the transcriptome analysis flowers of five red- (Red1-5) and five yellow-flowering (Yellow1-5) individual plants originated from the four wild type populations Point Loma, San Elijo, Potrero and Campo were chosen.  The floral samples were collected at the same time of the day and included two different developmental stages, flower buds measuring 3-5mm of visible petal tissue (Dev1) and mature flowers, which had opened the day before harvest (Dev2).  Plants were propagated in Zurich from seeds collected from natural populations.  All samples were frozen immediately in liquid nitrogen and stored at -80ºC. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Remaining genomic DNA was digested by RNase-free Turbo DNase (Thermo Fisher Scientific, MA, USA). The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, CA, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq RNA Sample Prep Kit v2 (Illumina, Inc., CA, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Caliper GX LabChip® GX (Caliper Life Sciences, Inc., USA). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10 nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20.