Rneasy plant mini extraction kit

RNA was extracted using the Rneasy plant mini extraction kit (Qiagen). The plant material in RNAlater was thawed just enough to remove approximately 20–30 mg. This was placed into a tube with a sterile stainless steel 6 mm bead. The tube, sample, bead and adapter set were then cooled at −80 °C for 30 min. After cooling, the plant tissue was disrupted by beating using the TissueLyser II (Qiagen) at 30 Hz for 2 min. The kit protocol was then followed for the remainder of the extraction. The RNA was eluted in 50 µl of sterile water. Extractions were pooled and concentrated as described above. Before concentrating, the pooled RNA was treated with DNase, using the rDNase Set (Macherey-Nagel).

Based on the phytochemical results, pre-stored pooled samples (two plants) of two genotypes (reflecting the greatest differences with other genotypes) from each clone (genotype) were selected for the RNA sequencing. A total of four samples (harvested at early blossoming stage and from flowering aerial parts of plants) were used for RNA isolation (Qiagen RNeasy plant mini extraction kit, QIAGEN, Hilden, Germany). Briefly, samples with high RNA quality and integrity were used for cDNA library construction (TruSeq standard mRNA preparation kit, Illumina company, CA, USA). The Illumina sequencing machine completed 150-cycle pair-ended sequencing at the end. The data were first inspected using FASTQC v0.11.9 [58 ] and then trimmed to remove low-quality bases (Q < 30) and adaptor contaminations using Trimmomatic v0.39 [59 (link)]. De novo assembly of the dataset was performed by utilizing an ensemble workflow that integrates EvidentialGene pipelines [60 ] in a concatenation method, as described in our recently published article [31 (link)]. The clustered, non-redundant, and final assembled transcripts of the EvidentialGene tool were called unigenes.

RNA was extracted according to Kruse et al. [39 (link), 41 ]. In brief, three biological replicates, one plate per replicate, were used for RNA-seq analysis. RNA was extracted using a RNeasy Plant Mini Extraction Kit (Qiagen) according to standard protocols. RNA integrity (RIN ≥ 8) was verified using an Agilent 2100 Bioanalyzer. RNA samples were sent to the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine, St Louis, MO. Paired-end sequence was obtained from an Illumina 2500 Hiseq (2 X 101 bp) using the Ribo-Zero rRNA Removal Kit prior to library preparation. Read pairs for each biological replicate were aligned to TAIR10 [43 (link), 44 (link)] assembly using Spliced Transcripts Alignment to a Reference (STAR) [45 (link)]. Gene level read counts were determined using HTSeq-count. For transcript/isoform analysis, Sailfish (v0.9.0) was used for splice aware transcript quantification [46 (link)]. Differential expression was determined using the generalized linear model likelihood test within the Empirical Analysis of Digital Gene Expression Data in R (EdgeR) package [47 (link), 48 (link)].