Oxford Nanopore MinION Sequencing

The ‘strand switching cDNA by ligation’ approach Library from total mRNA was prepared using the Ligation Sequencing kit (SQK-LSK108; Oxford Nanopore Technologies) following the 1D Strand switching cDNA by ligation protocol. Briefly: (ss)cDNA synthesis was carried out using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) and an anchored adapter-primer with (VN)T₂₀ nucleotides (nts). A 5′ adapter sequence with three O-methyl-guanine RNA bases was added for the facilitation of strand switching. PCR was carried out using Kapa HiFi DNA polymerase (Kapa Biosystems) and the primers supplied in the kit. End repair was conducted using NEBNext End repair/dA-tailing Module (New England Biolabs) followed by adapter ligation using adapters (supplied in the kit) and NEB Blunt/TA Ligase Master Mix (New England Biolabs). The cDNA sample was purified between each step using Agencourt AMPure XP magnetic beads (Beckman Coulter) and the library concentration was determined using a Qubit 2.0 Fluorometer through use of the Qubit (ds)DNA HS Assay Kit (Thermo Fisher Scientific). Samples were loaded on R9.4 SpotON Flow Cells, and base calling was performed using Albacore v1.2.6.

The Cap-selected mRNA sample was subjected to end repair and adapter ligation steps – as was described above – before loading on the Flow Cells.

The direct RNA sequencing approach Libraries were prepared using the Direct RNA Sequencing Kit (SQK-RNA001; Oxford Nanopore Technologies) The first strand cDNA was synthesized by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) using an RT adapter with T₁₀ nts. Adapters, supplied in the kit, were ligated using T4 DNA ligase (New England Biolabs). The RNA-DNA hybrid was purified between each step by using Agencourt AMPure XP magnetic beads (Beckman Coulter), treated with RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific). Sample concentration was determined using a Qubit 2.0 Fluorometer and Qubit DNA HS Assay Kit (Thermo Fisher Scientific). Libraries were loaded on R9.4 SpotON Flow Cells. Albacore software (v1.2.6) was used for base calling.