title: Oxford Nanopore MinION RNA-Seq of Cynara cardunculus var. altilis: mixed tissues
design: We modified and adapted the ONT Nanopore cDNA-PCR Sequencing Kit SQK-PCS108 (Oxford Nanopore Technologies, Oxford, UK) for the preparation of library starting from total RNA as reported in Picelli et al. (2013 and 2014). To ensure wide coverage of transcript isoforms, many of which may be tissue specific, we multiplexed all the sampled tissues and pooled them for subsequent sequencing. In brief, 600 ng of total RNA was denatured at 65 C for 5 min for Oligo-dT30VN hybridization, then reverse transcribed using strand-switching protocol in 20 l of reaction mixture as reported in the relevant cDNA-PCR Sequencing kit protocol (version: PCS_9035_v108_revF, Oxford Nanopore Technologies, Oxford, UK). Reaction was incubated at 42 C for 40 min then with 10 cycles of (50 C for 2 min, 42 C for 2 min) to optimize template-switching followed by 1 cycle of (80 C for 10 min) for enzyme inactivation. The double-stranded cDNA was split into four PCR reactions for which we used Long AmpTaq 2x MasterMix (NEB) and cDNA primer (cPrim) for amplification incubating at 95 C for 30 s followed by 20 cycles of (95 C for 15s, 50 C- 55 C for 15s, 65 C for 3 min), with a final extension at 65C for 6 min. Purification steps were carried out following manufacturers instructions and the resulting cDNA was evaluated and quantified using an Agilent High Sensitivity DNA kit and Chip on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) (SI Figure 2). Adapter (cAMX) was added to 23 l of amplified cDNA library then reaction was purified with 1x volume Agencourt AMPure XP beads (Beckman Coulter), according to ONT protocol. Purified library was quantified by fluorometric quantitation and 600 fmol was mixed with Library loading beads and Running buffer with fuel mix provided in ONT protocol. The full-length cDNA library was then sequenced on a MinION R9.5 flow cells for 48 h using 1D sequencing protocol.