2.5 RNA sequencing
The Sequencing Section, Okinawa Institute of Science and Technology
Graduate University, Japan carried out library preparation and
sequencing on all 40 RNA samples. RNA was quantified by Qubit Flex
Fluorometer (Qubit RNA BR assay kit, Thermo Fisher Scientific Inc.). The
NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs
Inc.) was followed according to the manufacturers protocol to isolate
mRNA. One library was prepared for each sample, using the NEBNext® Ultra
II Directional RNA Library Prep Kit for Illumina® (New England BioLabs
Inc.) following the manufacturers protocol, using ten PCR cycles.
Libraries were sequenced on two lanes of a NovaSeq6000 with a S2 flow
cell paired end to the length of 150 bp.