Illumina reads of cDNA library generated from 'Rush' leaf tissue
Prior to RNA extraction, Tissuelyser II adapters, safelock tubes, and 5mm stainless steel beads were chilled at -80C for 2 hours.  Working on dry ice, leaf and kernel tissue was added to prechilled tubes and disrupted using the Tissuelyser II for 2 rounds at 30Hz for 1 minute each round. Samples were then processed using the Qiagen Plant RNeasy with an on-column DNA digest.  Total RNA was assayed for purity and integrity using a NanoDrop One Spectrophotometer and Agilent 2100 Bioanalyzer, respectively.  RNA libraries were prepared from samples that met the TruSeq Stranded Total RNA Sample Preparation Guide (15031048 E) input guidelines using the Illumina TruSeq Stranded Total (Plant) RNA Sample Preparation kit (Illumina Inc., San Diego, California, USA).  For each library preparation, cytoplasmic, mitochondrial and chloroplast ribosomal RNA was removed using biotinylated target-specific oligos combined with paramagnetic beads tagged with streptavidin.  Following purification, the reduced RNA was fragmented using divalent cations under elevated temperature.  Fragmented RNA was copied into first stranded cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) and random primers.  Second strand cDNA was synthesized using a modified dNTP mix (dTTP replaced with dUTP), DNA Polymerase I, and RNase H.  Double-stranded cDNA was cleaned up with AMPure XP Beads (1X) (Agencourt, Beckman Coulter).  The cDNA products were incubated with Klenow DNA Polymerase to add a single A nucleotide to the 3 end of the blunt DNA fragments.  Unique dual indexes (UDI) were ligated to the DNA fragments and cleaned up with two rounds of AMPure XP beads (0.8X).  Adapter ligated DNA was amplified by PCR and cleaned up with AMPure XP beads (0.8X).  Final libraries were assessed for size and quantity using an Agilent DNA1000 chip and a Qubit dsDNA HS Assay Kit (Invitrogen, Carlsbad, California, USA), respectively.  Libraries were standardized to 2nM, and 150-bp paired-end sequencing was performed using standard GBS chemistry on an Illumina NovaSeq6000 sequencer.