Technology 2100 bioanalyzer

The total RNA samples from the bark region were prepared according to our previous work^{32 (link)} and outsourced to Macrogen Inc., Korea, for sequencing with Illumina TruSeq Stranded mRNA sample Prep kits after poly-A tailing enrichment. The prepared cDNA library size and concentration were determined using an Agilent Technology 2100 Bioanalyzer with D1000 screen tape. The library size ranged from 271 to 293 bp. RNA extracted from 8 plants (bark region) were pooled for each biological replicate. Two independent biological replicates were used for each experimental conditions (i.e. control, JA, LA, and ET) resulting in a total of eight prepared libraries. Four libraries each were multiplexed in a single lane of the Illumina sequencing run. 30% of the PhiX controls were added per sample to avoid bias of the GC content. The libraries were then sequenced using an Illumina Hiseq2500 sequencing system in rapid-run mode, which generated 150 bp paired-end reads. We targeted a high sequencing depth approach on the ssRNA-seq instead of targeting a higher number of biological replicates as greater sequencing depth would provide maximum power to detect more informational reads¹¹⁵ , including those with weak expressions, for the differential expression analysis. In addition, estimation accuracy for low expression genes is known to be improved by adding sequencing depth^{116 (link)}.

Shotgun sequencing libraries from the two adult samples and two technical replicates of one juvenile sample were prepared and multiplexed using the Illumina Nextera XT kit and protocol. Input DNA was quantified using the Qubit dsDNA system. Libraries were checked using the Agilent Technology 2100 Bioanalyzer. Libraries were manually normalized due to a final library yield under 10 nM. Paired-end sequencing was performed on an Illumina MiSeq using a MiSeq Reagent Kit V2.