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.