Hiseq 2500 flow cell

RNA was harvested from each of the four samples (row 2 of Figure 1). The four RNA samples were then processed twice on different days using the Agilent Quick Amp Labeling kit (Part no. 5190-0424) to produce eight cRNA libraries, each of which was then hybridized on two separate chips (Yeast Expression 8 × 15K arrays) on different days according to the factorial design. After the arrays were washed and scanned, features were extracted using the Agilent Feature Extraction software to determine red and green intensities.
The same four RNA samples were also processed using the Illumina TruSeq RNA Sample Prep v2 LS protocol. Each sample was prepared twice up to the 3′ end adenylation step, then each of the eight preparations was split into two aliquots, after which each was indexed and amplified to complete the RNA-Seq library preparation (row 3 of Figure 1). The two groups of eight samples that were indexed together were each mixed at equimolar concentrations and sequenced on separate lanes on the same Illumina HiSeq 2500 flowcell, to produce 141 bp reads. In total, we produced profiles from 16 RNA-Seq samples and 16 microarray samples with identically nested experimental designs (row 4 of Figure 1).

RNA (~3 μg per sample) was used as the input material for constructing libraries. RNA-seq libraries were prepared using the TruSeq Paired-End (PE) Cluster Kit v3-cBot-HS (Illumina, PE125) according to the manufacturer's protocol. Libraries from fertile and sterile flowers, with three biological replicates, were sequenced in a single Illumina Hiseq 2500 flowcell, generating >139 million paired-end reads per sample. A Perl script was written to remove low-quality sequences (reads with a base quality < 20). For de novo reference transcriptome assembly, all high-quality RNA-Seq reads were pooled from the Illumina sequencing of each of the six samples (three biological replicates) and were then used as input for assembly using Trinity software (Grabherr et al., 2011 (link)). All raw sequence data have been deposited in the NCBI Sequence Read Archive (SRA, accession number SRP076665).

The GBS approach [11 (link)] was used to discover SNP markers. The mapping population was a set of 213 (of the 275) F₁ individuals of the progeny TGdL x MB, including the 163 plants previously analysed by Beltramo et al. (2016) and 50 new individuals [29 ]. Only the plants situated at the edge of the experimental field were excluded.
Total genomic DNA was extracted from young leaves collected in the spring using a CTAB method [31 ]. Quantity and quality of extracted DNA were determined by the Qubit assay (Thermo Fisher Scientific) and by electrophoresis on 1% agarose gel and comparison against a Lambda DNA/EcoRI + HindIII marker (Thermo Fisher Scientific). In October 2014, approximately 3 μg of genomic DNA from each individual and the two parents were sent to the Genomic Diversity Facility at Cornell University—Institute of Biotechnology (USA) (http://www.biotech.cornell.edu/brc/genomic-diversity-facility) for GBS. Briefly, GBS libraries were constructed in 96-plex by digesting genomic DNA with the restriction enzyme ApeKI, a five-base cutter (5’ GCWGC 3’), followed by ligation of a barcode adaptor and a common Illumina sequencing adaptor to the fragmented DNA. The resulting libraries were run through an Illumina Hiseq2500 flow cell for sequencing.