Rnase cocktail enzyme mix

A standard PURE reaction programmed with 17.5 nM (250 ng) of a linear Rep DNA template (under control of a T7 promoter) was supplemented with 0.6 µl FluoroTect™ Green_Lys tRNA (FluoroTect™ Green_Lysin vitro translation labelling system, Promega). Template DNA was omitted in the negative control reaction. Samples were incubated for 2  h at 37 °C in a nuclease-free PCR tube (Thermo Fisher Scientific) using a ProFlex PCR System (Thermo Fisher Scientific) and subsequently treated with 0.6 μl RNase Cocktail™ Enzyme Mix (0.5 U/μl RNase A and 20 U/μl RNase T1, Thermo Fisher Scientific) for 15 min at 37°C to degrade non-incorporated Green_Lys tRNA. 7.5 μl sample were mixed with an equal volume 2× Laemmli sample loading buffer (incl. 200 mM DTT) and denatured for 2.5 min at 65°C. Samples were analysed by conventional discontinuous SDS-PAGE (10% gel) run at 4°C (100 V for 10 min, then 200 V) on a Midi-format electrophoresis system (Atto). The fluorescent signal of the de novo expressed rep β subunit was imaged on a fluorescence laser scanner (Typhoon FLA 9000, GE Healthcare) at either 473 nm (blue LD laser/510LP filter) or at 532 nm (green SHG laser/575LP filter). Total protein and the molecular-weight size marker (PageRuler™ Unstained Protein Ladder, Thermo Fisher Scientific) were visualized after SYPRO Ruby (Bio-Rad) staining using the same instrument (473 nm, blue LD laser/575LP filter).

Direct cDNA libraries were prepared from the mock and six BoHV-1 p.i samples in three replicates using the ONT’s Direct cDNA Sequencing Kit (SQK-DCS109) according to the manufacturer’s instructions. The first cDNA strand synthesis was performed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) with SSP and VN primers (supplied in the kit) and 100 ng of poly(A) + RNA for each sample. This was followed by the removal of potential RNA contamination using RNase Cocktail Enzyme Mix (Thermo Fisher Scientific), and second strand synthesis using LongAmp Taq Master Mix (New England Biolabs). Double stranded cDNA ends were repaired using NEBNext End repair /dA-tailing Module (New England Biolabs). This was followed by ligation of sequencing adapter employing the NEB Blunt /TA Ligase Master Mix (New England Biolabs). Libraries were barcoded using Native Barcoding (12) Kit (ONT) according to the manufacturer’s instructions.

The freshly nebulized vaccine samples and stock vaccine samples (control condition) were split into two equal volumes of 100 µl. One subsample was treated for 5 min at 37 °C with RNase Cocktail Enzyme Mix (Thermo Fisher Scientific), containing a combination of RNaseA and RNaseT1 at a final concentration of 5 and 200 U/ml, respectively. This RNase treatment will completely degrade any vaccine mRNA that is not protected by intact LNP. After the RNase treatment, 300 μl Trizol LS reagent (Thermo Fisher Scientific) was added to the mixture to inactivate the RNases.

ONT Direct cDNA Sequencing Kit (SQK-DCS109) was used for the generation of amplification-free libraries. In short, 100 ng polyA-selected RNA sample was used for the synthesis of the first cDNA strand using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) RNase Cocktail Enzyme Mix (Thermo Fisher Scientific) was used for the removal of RNAs from the single stranded cDNA molecules. The synthesis of the second cDNA strand was performed using LongAmp Taq Master Mix (New England Biolabs). cDNA ends were repaired using NEBNext Ultra II End Repair/dA-Tailing Module. The cDNA ends were repaired using NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs). Libraries were barcoded using ONT Native Barcoding Expansion Kit (EXP-NBD104), then the ligation of the sequencing adapter was carried out using NEB Quick T4 DNA Ligase. All conditions were set according to the SQK-DCS109 manufacturer’s protocol.

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).