2.3 | Library preparation for Illumina sequencing
The sequencing library was prepared using a NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770) together with a NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext® Multiplex Oligos for Illumina (E6440) following the manufacturer’s protocol (New England BioLabs, Massachusetts, USA). One microgram of the total RNA was used to start the library preparation. The final PCR enrichment was performed for eight cycles. After the purification of the enriched product’s using 0.9× Agencourt AMPure XP, equal amounts of products were pooled from all libraries and sequenced on an Illumina MiSeq with paired-end reads with 300 cycles. Sequencing was done at the NGS High Throughput Genomics Core of the Biodiversity Research Centre, Academia Sinica, Taiwan.