RNAseq of Distal region of Stage 2 petal of Hibiscus trionum, plant 5, forward reads
Tissue was collected from five individual H. trionum CUBG plants to generate five biological replicates: for each biological replicate, stage 2 petals were harvested and dissected to separate proximal, boundary and distal region. Tissue from these 15 samples were flash frozen in liquid nitrogen, ground to a fine powder, and total RNA extracted using the Spectrum Plant Total RNA kit (Sigma STRN250) with 40 mg of starting material. An on-column DNase I (Sigma DNASE70) treatment was performed between the first two column washes. RNA was eluted with 30 l nuclease-free H2O (Promega MC1191). RNA purity was analyzed using a NanoDrop (ThermoFisher Scientific) and RNA ScreenTape (Agilent 5067-5576, 5067-5577, 5067-5578) on the Agilent TapeStation 2200 system. Samples with minimum 1.75 g total RNA (>50 ng.l-1), and A260/280 value of 2.0 were shipped on dry ice to Azenta Life Sciences, Leipzig (formerly GeneWiz) for RNA-seq library preparation and sequencing. Library preparation was performed with polyA selection and sequencing using an Illumina HiSeq machine (2x 150bp configuration, ~350M raw paired-end reads per lane, at least 30M reads per sample).