Nebnext ultra rna library prep kit for v1

cDNA library preparation and Illumina MiSeq sequencing were conducted following previously established protocols [20 (link)]. Briefly, individual strains were subjected to library construction using a NEBNext Ultra RNA library Prep Kit for Illumina v1.2 (New England Biolabs, Ipswich, MA, USA), incorporating bar-coded adapters to generate a 200 bp fragment library. The resulting libraries were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) and were assessed for quality on a MultiNA MCE-202 bioanalyzer (Shimadzu Corporation, Kyoto, Japan). Nucleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina, San Francisco, CA, USA) with a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was carried out with CLC Genomics Workbench v22 (CLC Bio, Tokyo, Japan). The complete or nearly complete nucleotide sequence of each gene segment of Ghanaian RVA strains was obtained by de novo assembly and mapping reads to reference in CLC Genomics Workbench.

The extracted RNA was subjected to Illumina MiSeq NGS as described previously [36 (link)]. In brief, a 200 bp fragment library ligated with bar-coded adapters was constructed for 14 NoV GII strains using an NEBNext Ultra RNA Library Prep Kit for Illumina v 1.2 (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. A cDNA library was isolated using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). After evaluating the quality and quantity of the isolated cDNA library, 151-cycle paired-ends-read nucleotide sequencing was performed on a MiSeq Reagent Kit v2 (Illumina Inc., San Diego, CA, USA). MiSeq sequence data were analyzed using CLC Genomics Workbench v8.0.1 (CLC Bio, Aarhus, Denmark). Contigs were assembled from the obtained sequence data (trimmed) using de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) non-redundant nucleotide database was used to determine which contigs represented full-length nucleotide sequences for each gene segment of the 14 NoV strains. To further refine the contigs, sequence reads for each gene segment were mapped back to the assembled contig. The nucleotide sequences of the strains were translated into aa sequences using GENETYX v11 (GENETYX, Tokyo, Japan)

Viral RNA was extracted from 10∼20% faecal suspensions in PBS using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions. To assess the integrity of RVA dsRNA, polyacrylamide gel electrophoresis (PAGE) was carried out at constant current (30 mA per gel) on a precast 10% ePAGEL (ATTO Corporation, Tokyo, Japan). RVA dsRNA bands were visualized by SYBR Gold staining.
Using the Qubit RNA Assay, sample RNA concentrations were determined on a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), and starting concentrations normalized to 10∼100 ng for library construction. A 200 bp fragment library was constructed for each sample using the NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs, Ipswich, MA, USA) according to manufacturer's instructions. Samples were bar-coded for multiplexing using NEBNext Multiplex Oligos for Illumina, Index Primer Sets 1 and 2 (New England Biolabs, Ipswich, MA, USA). Library purification was done using Agencourt AMPure XP magnetic beads (Beckman Coulter, Pasadena, CA, USA) as recommended in the NEBNext protocol. The quality of the purified libraries was assessed on a MultiNA MCE-202 bioanalyzer (Shimadzu Corporation, Kyoto, Japan) and the concentrations determined on a Qubit 2.0 flourometer using the Qubit HS DNA Assay (Invitrogen, Carlsbad, CA, USA).

RNA extraction from foliage of three CLB⁺ and three CLB^- seedlings was done using a modified version of the protocol of Rajakani et al. (2013) (link) (see section 1.4 of the Supplementary Methods for details). mRNA enrichment was done using protocol C of the Thermo Scientific™ MagJET mRNA Enrichment Kit (Life Technologies Inc., Burlington ON, Canada). Libraries were made using the NEB Next^® Ultra™ RNA Library Prep Kit for Illumina^® v. 1.2. (New England BioLabs^® Inc., Ipswich MA, USA). DNA was purified as required using the Thermo Scientific GeneJET NGS Cleanup Kit (Life Technologies Inc.), and size selection (∼450 bp fragment size) was completed with the Thermo Scientific MagJET NGS Cleanup and Size Selection Kit (Life Technologies Inc.). Libraries were barcoded using the NEB Next^® Multiplex Oligos for Illumina^® - Index Primers Set 1 (New England BioLabs^® Inc.). Quality control and quantification of the individual libraries was done with a DNA 1K Analysis Kit (Bio-Rad Laboratories, Mississauga ON, Canada) in an Experion™ Automated Electrophoresis Station (Bio-Rad Laboratories). The final pool consisted of 40 ng of DNA per library. Pair-ended 100 base sequencing was completed in a single lane of an Illumina^® HiSeq 2000 sequencer at Genome Quebec Innovation Centre (Montreal QC, Canada).

Preparation of a cDNA library and Illumina MiSeq sequencing were performed as described previously [9 (link), 16 (link)–18 (link)]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for strains PCB-180, SKT-109, and SSKT-41 using NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (New England Biolabs) according to the manufacturer’s instructions. Library purification was carried out using Agencourt AMPure XP magnetic beads (Beckman Coulter). The quality of the purified cDNA library was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Nucleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was performed using CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) non-redundant nucleotide database was searched to obtain the full-length nucleotide sequence of each gene segment of strains PCB-180, SKT-109, and SSKT-41. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).

Building of a cDNA library and Illumina MiSeq sequencing were performed as described previously [14 (link), 16 (link), 30 (link), 33 (link), 34 (link)]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for the 14 strains using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs), and NEBNext Multiplex Oligos for Illumina (New England Biolabs) according to the manufacturer’s instructions. The cDNA library was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). After assessing the quality and quantity of the purified cDNA library, nucleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was performed using CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads (trimmed) by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) non-redundant nucleotide database was searched to obtain the full-length nucleotide sequence of each gene segment of the 14 strains. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).