RNA sequencing was performed for a total of 15 samples (four for FF, five for MF and six for IMF) through a paid service at the DNA Sequencing Core Facility of The University of British Columbia (Vancouver, BC, Canada), using the Illumina NextSeq 500 via Paired-End (2 × 42 bp reads). All raw reads of these libraries have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under the BioProject accession PRJNA669389, BioSample accessions SAMN16447877–SAMN16447891, and SRA accessions SRR12831863–SRR12831877.

Raw RNA-Seq reads were processed using CLC Genomics Workbench v11.0 (Qiagen) for removing low-quality and short reads, trimming adaptors and generating paired reads. The resulting sets of paired reads were then assembled separately with respect to FF, MF and IMF using the CLC Genomics Workbench with a word size of 60. The three assemblies were combined and cleaned up (by removing the redundant sequences) using CD-HIT-EST (Li and Godzik 2006) with the threshold of 98% identity to generate a final reference transcriptome sequences. The transcriptome was validated by mapping the preprocessed paired reads back to the reference assembled sequences using CLC Genomics Workbench. The de novo-assembled reference transcript was then BLAST’ed against UniProKB database using tBLASTx with an e-value threshold of 0.001. We also BLAST’ed the assembled transcripts against Purple Kush reference transcriptome (van Bakel et al. 2011) under the same setup for further validation. The resulting UniProKB BLAST output was annotated and mapped with GO terms using the Blast2GO PRO plug-in for CLC Genomics Workbench.

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