4.4. Oxford Nanopore Technologies Nanopore Sequencing Using the MinION Device

The Direct RNA sequencing (SQK-RNA002) protocol (Version: DRS_9080_v2_revM_14Aug2019) was used to obtain amplification-free transcriptomic data to remove RT and PCR biases, as well as to explore attributes of native RNA such as modified bases. Five hundred nanograms of Poly(A)+-tailed RNA was used. The library preparation was carried out according our previous publication [51] with the following modification: Agencourt RNAClean XP beads (Beckman Coulter, Brea, CA, United States) was used instead of the RNase OUT (Invitrogen)-treated Agencourt XP beads (Beckman Coulter, Brea, CA, USA).

Non-amplified cDNA libraries were prepared from the poly(A)+ fraction of RNAs from the MdBio strain using the ONT’s Direct cDNA Sequencing Kit (SQK-DCS109; DCS_9090_v109_revJ_14Aug2019, Oxford Nanopore Technologies, Oxford, United Kingdom) according to the manufacturer’s protocol. In brief, the Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, United States) with SSP and VN primers (supplied in the kit) were used for the synthesis of first cDNA strand from 100 ng of poly(A)+ RNA. Next, the potential RNA contamination was eliminated using RNase Cocktail Enzyme Mix (Thermo Fisher Scientific, Waltham, MA, United States). This step was followed by the second strand synthesis using LongAmp Taq Master Mix (New England Biolabs, Ipswich, MA, United States). Double-stranded cDNA ends were repaired using NEBNext End repair/dA-tailing Module (New England Biolabs, Ipswich, MA, United States), then the sequencing adapter ligation was carried out with the NEB Blunt/TA Ligase Master Mix (New England Biolabs).

ONT’s ligation-based sequencing protocol (SQK-LSK109; Version: GDE_9063_v109_revU_14Aug2019).

The ONT’s LSK109 protocol was used for sequencing the Poly(A)-selected oligo(dT)-primed, rRNA-depleted random-primed, or Terminator^TM-handled oligo(dT)-primed samples. The usefulness of the application of Terminator^TM enzyme is that it enriches the capped full-length transcripts, because this enzyme processively digests the RNA molecules with 5´-monophosphate ends but not with 5´-triphosphate, 5´-cap or 5´-hydroxyl groups.

The generation of cDNA was conducted according to our previous publications [31,51] using oligo(dT) or random primers. The DNA repair was carried out according to the SQK-LSK109 protocol. Briefly, the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair/dA-tailing Module reagents (all from New England Biolabs, Ipswich, MA, United States) were mixed with the samples, then the mixtures were incubated at 20 °C for 5 min and at 65 °C for 5 min. This step was followed by the adapter ligation: The NEBNext Quick T4 DNA Ligase (New England Biolabs), the Ligation Buffer, and Adapter Mix (both from ONT’s Kit, ONT, Oxford, United Kingdom) were mixed with the cDNA samples and incubated for 10 min at room temperature. Samples were purified using the AMPure XP magnetic beads (Beckman Coulter, Brea, CA, United States) after each enzymatic step.

1D Strand switching cDNA by ligation method (Version: SSE_9011_v108_revS_18Oct2016) and the ONT Ligation Sequencing Kit 1D (SQK-LSK108) (ONT, Oxford, United Kingdom)

This protocol was used to analyze the random primed cDNA libraries. In short, ribodepleted RNA fraction was used to generate cDNA samples, first it was mixed with dNTPs (10 mM, Thermo Scientific) and random primers (ordered from IDT DNA) and then the mixtures were incubated at 65 °C for 5 min. After this step, the DTT and buffer form the SuperScipt IV Reverse Transcriptase kit (Life Technologies), RNase OUT enzyme (Life Technologies), and strand-switching oligo with three O-methyl-guanine RNA bases (PCR_Sw_mod_3G; Bio Basic, Canada) were added and the mixtures were heated to 42 °C for 2 min. SuperScript IV Reverse Transcriptase enzyme (200 unit) was mixed into the samples. The generation of the first cDNA strand was conducted at 50 °C for 10 min, then the strand switching step was carried out at 42 °C for 10 min. For the inactivation of the enzymes, the samples were heated to 80 °C for 10min. Samples were amplified using the KAPA HiFi DNA Polymerase (Kapa Biosystems, Wilmington, MA, USA) and Ligation Sequencing Kit Primer Mix (provided by the 1D Kit, ONT, Oxford, UK). The NEBNext End repair/dA-tailing Module (New England Biolabs, Ipswich, MA, USA) was applied to repair cDNA ends, while NEB Blunt/TA Ligase Master Mix (New England Biolabs, Ipswich, MA, USA) was used to ligate the adapters (supplied by the kit, New England Biolabs, Ipswich, MA, USA).