4.3. Illumina Library Construction, Sequencing, and De Novo Assembly

The Illumina library for each organ was constructed using a NEBNext Ultra™ RNA Library Prep Kit (NEB, Ipswich, MA, USA), according to the manufacturer’s protocol. Sequencing was performed on the Illumina NovaSeq platform, generating paired-end (PE) reads with lengths of 150 bp. Illumina RNA-Seq raw reads were processed using in-house Perl scripts. Clean reads were obtained by removing the adapter reads, reads with more than 10% ambiguous bases ‘N’, and low-quality reads (Qphred ≤ 20 base with more than 50%) from the raw reads. Clean reads were de novo assembled with Trinity v.2.4.0 [109] using the following parameters: min_kmer_cov: 2, as well as the remaining default parameters.

The Illumina RNA-Seq transcriptome raw data were deposited in the SRA of NCBI as follows: root: SRR11715801; stem: SRR11715800; leaf: SRR11715799; strobilus: SRR11715798.