Sampling strategies for sugarcane using either clonal replicates or diverse genotypes can bias the conclusions of RNA-seq studies

We collected immature internodes from a diverse set of sugarcane genotypes present in the Brazilian Panel of Sugarcane Genotypes, which have a broad range of soluble solids content (ºBrix). The twelve varieties we selected have representants of S. officinarum, S. spontaneum, and commercial hybrids. We organized the genotypes into four groups according to their soluble solids content level, namely Very Low, Low, High, and Very High ºBrix (VLB, LB, HB, and VHB). The immature culms (internode +1) were collected in June 2016, in Araras (Brazil), followed by extraction of total RNA with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's recommendations. We prepared the RNA-Seq libraries of polyadenylated transcripts using the TruSeq Stranded mRNA LT (Illumina) protocol. These libraries were sequenced in a HiSeq 2500 equipment (Illumina), resulting in paired-end reads 2x100 bp long. We used a single lane exclusively for all samples.