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.