Library Preparation and Direct RNA Sequencing by Nanopore

Direct RNA sequencing was performed using the Direct RNA Sequencing Protocol (SQK-RNA001 kit) for the MinION. Some steps of the manufacture protocol were modified. First, the ligation reaction of RNA and adaptor was extended from 10 to 15 min. At this step, control RNA (RCS) was not added. Second, the RT (50 min) step was skipped in our protocol. Third, wire-bored tips (Axygen) were used for mixing AMPureXP beads (Beckman Coulter)-RNA. RNA library was eluted and loaded onto a flow cell for sequencing. Sequencing of the RNA was performed on a single R9.4/FLO-MIN106 flow cell on a MinION Mk1B for ∼8 h.