Tissue sampling, RNA extraction, and sequencing

Tissues from 5 stages of flowering and “fruit” development were harvested from untreated flowers in biological duplicates or triplicates for RNA isolation. The stages of flowering followed those identified in Fragaria by Kang et al. [12], with the addition of a stage 0 (unopened flowers) and young unexpanded leaf tissue. The selected developmental stages are shown in Fig. 3. RNA was extracted from 50 mg of snap-frozen tissue from each developmental stage using the Spectrum plant total RNA extraction kit (Sigma) with an on-column DNase I digestion (Sigma) step. The extraction protocol followed the manufacturers’ recommendations with 2 minor modifications: 1% Polyvinylpyrrolidone (PVP) was added to the lysis solution and the number of washes at each stage was doubled (ie, 2 washes were performed with wash solution 1 and 4 washes were performed with wash solution 2). The RNA extracted from each sample was diluted in 50 μL of elution solution (Sigma). Following elution, total RNA was quantified using a Nanodrop spectrophotometer and Qubit fluorometer and assessed for integrity using a Bioanalyzer (Agilent). Samples returning a RNA integrity number (RIN) value greater than 7.5 were considered acceptable for sequencing. A total of 12 Illumina TruSeq libraries were constructed from 2 μg of total RNA. Libraries were made from the following samples: 1 from stage 0, 2 from stage 1, 2 from stage 2, 3 from stage 3, and 3 from stage 4. A final library was made from RNA of young leaf tissue. The libraries were sequenced in triplex per single lane of Illumina HiSeq2000. Samples were indexed and multiplexed, then 101 bp paired-end sequencing was performed using the Illumina HiSeq 2000 platform at the Weill Medical core genomics facility at Cornell University.