Identification of PawS1 and PawS-Like (PawL) genes in Heliopsis helianthoides seeds by high-throughput sequencing
RNA extraction: Mature dry H. helianthoides seeds were purchased from the Jelitto seed company, Germany. 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 DNase treated followed by further purification 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.