Longamp taq master mix

ONT’s direct (d)cDNA Sequencing Kit (SQK-DCS109) and the dcDNA protocol (ONT) was used to generate libraries from the poly(A)+ RNA samples (100 ng from each) according to the manufacturer’s recommendations. First, a reverse transcription step was carried out using Maxima H Minus Reverse Transcriptase enzyme (Thermo Fisher Scientific) and SSP and VN primers (supplied in the ONT kit). This step was followed by the removal of the potential RNA using RNase Cocktail Enzyme Mix (Thermo Fisher Scientific). For the synthesis of the second cDNA strand, LongAmp Taq Master Mix (New England Biolabs) was used. The end-repair was carried out using NEBNext Ultra II End repair/dA-tailing Module (New England Biolabs) and was followed by the adapter (AMX) ligation using NEB Blunt/TA Ligase Master Mix (New England Biolabs). Each library was barcoded using Native Barcoding Kit (ONT) as described in the manual (Table 1). Mock-infected samples and libraries from the earlier time points were run separately from the later time points in order to avoid the potential “barcode hopping”. Agencourt AMPure XP magnetic beads (Beckman Coulter) were used for purification of the samples following each enzymatic step of the protocol. The concentrations of the cDNAs and dcDNA libraries were measured using Qubit 4.0 and the Qubit dsDNA HS Assay Kit (Invitrogen).

Twenty-microgram aliquots of total RNA were diluted in 100 μl of nuclease-free water, and poly-A was selected using NEXTflex Poly(A) Beads (BIOO Scientific, catalog no. NOVA-512980). Resulting poly-A RNA was quantified, and 50-ng aliquots were transferred to thin-walled Eppendorf polymerase chain reaction (PCR) tubes. The biological poly-A RNA and a synthetic control (Lexogen SIRV Set 3, 0.5 ng) were prepared for cDNA synthesis and nanopore sequencing following the ONT SQK-PCS108 kit protocol with a few exceptions. During the reverse transcription step, Superscript IV was used, and the reverse transcription incubation time was increased to 15 min. After reverse transcription, PCR was performed using LongAmp Taq Master Mix (NEB) under the following conditions: 95°C for 30 s, 10 cycles (95°C for 15 s, 62°C for 15 s, and 65°C for 15 min) and 65°C for 15 min, hold at 4°C. The resulting cDNA libraries were quantified, and sequencing libraries were prepared using 700 ng of cDNA following the standard protocol for SQK-PCS108 (one-dimensional sequencing). Sequencing was performed on the GridION platform using ONT R9.4 flow cells and the standard MinKNOW protocol script (NC_48hr_sequencing_FLO-MIN106_SCK-PCS108).