Library Preparation for Nanopore Sequencing

The dRNA-Seq method using a direct RNA sequencing kit (SQK-RNA002, Version: DRS_9080_v2_revK_14Aug2019) from ONT was used for amplification-free sequencing. This approach is highly recommended to explore special features of native RNA (e.g., modified bases) and avoid potential biases associated with reverse transcription (RT) or PCR. Total RNA from eight samples (two parallel experiments from 4, 8, 12, and 20 h p.i.) was mixed together, and then the polyA(+) fraction of RNA was purified from the sample mix. One hundred nanograms from the polyA-tailed RNA were mixed with RT Adapter (oligo dT-containing T10 adapter), RNA CS (both from the Nanopore kit; the latter was used to monitor the sequencing quality), NEBNext Quick Ligation Reaction Buffer, and T4 DNA ligase [both from New England Biolabs (NEB)]. The mixture was incubated for 10 min at room temperature, and then RT was conducted to generate first-strand cDNA using dNTPs (NEB), 5 × first-strand buffer, DTT (both from Invitrogen SuperScript III) and UltraPure™ DNase/RNase-Free distilled water (Invitrogen), and then the sample was mixed with SuperScript III enzyme (Thermo Fisher Scientific). RT was performed in a Veriti cycler (Applied Biosystems) at 50°C for 50 min, and the reaction was subsequently terminated at 70°C for 10 min. RNA-cDNA hybrids were purified using Agencourt RNAClean XP Beads [1.8 bead ratio (BR)]; Beckman Coulter], washed with freshly prepared 70% ethanol, and eluted in UltraPure™ nuclease-free water. The sample was then ligated to the RNA adapter (RMX from the ONT kit) at room temperature for 10 min using NEBNext Quick Ligation Reaction Buffer, T4 DNA ligase, and nuclease-free water. The ligation reaction was followed by a final purification step using XP Beads (1.0 BR). Samples were washed with wash buffer (ONT) and eluted in elution buffer (ONT). After Qubit measurement, 100 fmol from the library were loaded onto a Flow Cell.

Viral and host transcripts were also sequenced on a MinION sequencer following the instructions of the direct cDNA sequencing kit (SQK-DCS109; Version: DCS_9090_v109_revJ_14Aug2019; ONT). This protocol is based on strand switching, and it is highly optimal for the generation of full-length cDNA for the identification of potential novel transcript isoforms without potential PCR bias. The starting material was 100 ng of a poly(A)+ RNA mixture from various time points of infection (4, 8, 12, and 20 h p.i.). An oligo dT-containing VN primer (VNP) and dNTPs (10 μM) were added to the RNA. After 5 min of incubation at 65°C, the following components were added: 5 × RT buffer (from the Maxima H Minus Reverse Transcriptase kit, Thermo Fisher Scientific), RNaseOUT™ (Thermo Fisher Scientific), strand switching primer from the ONT kit and nuclease-free water. This mixture was pre-heated at 42°C for 2 min, and Maxima H Minus Reverse Transcriptase was added. RT was conducted at 42°C for 90 min, and finally, the reaction was stopped by incubation at 85°C for 5 min. RNase Cocktail Enzyme Mix (Thermo Fisher Scientific) was used to degrade the RNA in the sample. Incubation was performed at 37°C for 10 min. Before the second-strand synthesis, the sample was cleaned using AMPure XP Beads (0.8 BR) (Beckman Coulter). Then, LongAmp Taq Master Mix (NEB) was used to synthesize second-strand cDNA using the PR2 primer (ONT). Samples were incubated using the following “only one cycle protocol:” denaturation at 94°C for min, annealing at 50°C for 1 min, and elongation at 65°C for 15 min. The double-stranded cDNAs were purified using AMPure XP method (0.8 BR). NEBNext Ultra II End-prep reaction buffer and NEBNext Ultra II End-prep enzyme mix (both from NEB) were added to each sample. This end repair process was performed at 20°C for 5 min, followed by a 5-min incubation at 65°C. Enzymes and buffers were removed from the reaction using the AMPure XP purification method (1.0 BR), and then the sample was subjected to adapter ligation. The ONT Adapter Mix and Blunt/TA Ligation Master Mix (NEB) were mixed with each sample, and the mixture was incubated at room temperature for 10 min. A final AMPure XP purification (0.4 BR) was conducted to remove any excess proteins, nucleotides, and salts from the DNA library. Adapter Bead Binding Buffer was used to wash the beads, and the library was eluted using elution buffer (both from the Nanopore kit). The samples were quantified using Qubit, and then 200 fmol from the sample were loaded on two MinION Flow Cells.

Samples from each time point (4, 8, 12, 20 h p.i. and the mock infected cells) were sequenced by the ONT MinION device using the cDNA-PCR Barcoding protocol (SQK-PCS109 and SQK-PBK004; Version: PCSB_9086_v109_revK_14Aug2019). This protocol is recommended to identify and quantify full-length transcripts, discover novel isoforms, and splice variants and fusion transcripts from a low amount of starting material (total RNA) to generate large amounts of cDNA data. Approximately 50 ng of each of the samples were used for library preparation. VNP and dNTPs were added to the RNA and incubated at 65°C for 5 min. The strand-switching buffer mixture (RT buffer, RNaseOUT, nuclease-free water, and SSP) was added to the samples, which were incubated at 40°C for 2 min. RT was conducted by adding Maxima H Minus Reverse Transcriptase at 42°C for 90 min. The enzyme was inactivated by increasing the temperature to 85°C for 5 min. LongAmp Taq Master Mix, one of the Low Input barcode primers (LWB01-12, from the ONT's SQK-PBK004 kit, Supplementary Table 4), and nuclease-free water were included in the RT reaction mixture. Supplementary Table 5 shows the PCR conditions. PCR products were treated with exonuclease (NEB), and the mixture was then incubated at 37°C for 15 min, followed by 80°C for 15 min. AMPure Beads (0.8 BR) was used for purification, and the clean sample was eluted.