Identification of PawS1 and PawS-Like (PawL) genes in Zinnia haageana seeds by high-throughput sequencing
RNA Extraction: Mature dry Z. haageana seeds were purchased from the B&T World Seeds company, France. RNA was extracted from whole seeds using the hot phenol method as described in Mylne et al., 2012, Plant Cell 24: 2765-2778. The total RNA solution was purified further using the NucleoSpin RNA Clean-up kit (Machery-Nagel). The quality of the RNA was tested by measuring the A260/280 and A260/230 ratios using a NanoDrop Spectrophotometer (ND-1000 Spectrophotometer, ThermoFisher Scientific) and visualizing on a 1% agarose gel stained with ethidium bromide.   Library construction: Sequencing libraries were generated using the TruSeq RNA sample prep kit (Illumina). To do so, 300-1000 ng of total mRNA was used according to the manufacturer’s instructions.  Sequencing was then performed on an Illumina HiSeq 1500, as a 2 X 101 paired-end run.    Data processing: FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) was used for quality trimming and filtering. Raw reads were trimmed using the FASTQ quality trimmer tool to obtain a quality threshold of the read ends of 30 by maintaining the minimum length after trimming at 50. FASTQ quality filter tool was used to remove poor quality reads to maintain a quality threshold at 22 and minimum percentage of bases that match the quality threshold 22 as 90.