title: TWISTEnrichedLibrary of  S.murinus kidney tissue #13
design: Kidney homogenates that were positive for novel orthonairovirus were centrifuged at 13,000 rpm for 1 min. RNA from the supernatant was extracted using a Direct-zol RNA miniprep (Zymo Research Corporation) following manufacturers instructions. An enriched NGS library was prepared from the tick sample using the Comprehensive Viral Research Panel (Twist Bioscience, San Francisco, CA, USA) coupled with a custom probe set designed from the three complete nairovirus segments detected in S. murinus. The Twist EF library prep kit (Twist Bioscience, San Francisco, CA, USA) was used to prepare an Illumina compatible NGS library, with all reagents supplied from the kit, unless specified. A cDNA template was synthesized from 50ng of tick RNA by firstly incubating the RNA with 50M Random Primer 6 (New England BioLabs) at 95C for 5 mins and cooling to 20C with a ramp rate of 0.1-0.2C per second. First strand cDNA was subsequently synthesized from the previous step in a 50l reaction consisting of 20l RNA-Random Primer 6 mix, 25l ProtoScript II Reaction Mix and 5l ProtoScript II Enzyme Mix (ProtoScript II First Strand cDNA Synthesis Kit, New England BioLabs) at 25C for 5 mins, 42C for 1 hr, 80C for 5 mins and a hold at 4C. Second strand cDNA was synthesized from the first strand in a 80l reaction consisting of 18l Nuclease-free water, 8l Second Strand Synthesis Reaction Buffer, 4l Second Strand Synthesis Enzyme Mix (NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module, New England BioLabs) at 16C for 1 hr. The tick cDNA template was purified with 1.2X DNA Purification beads with two 80% ethanol washes and elution with 25l Nuclease-free water, with a subsequent dilution to 1ng/l. Fragmentation, end repair and dA-trailing was carried out in a 50l reaction consisting of 25l DNA, 10l Nuclease-free water, 5l 10X Fragmentation Buffer and 10l 5X Fragmentation Enzyme at the following conditions of 32C for 22 mins, 65C for 30 mins and a hold at 4C. Adapters were ligated to the cDNA in a 50l reaction containing 2.5l Twist Universal Adapters, 17.5l Nuclease-free water, 20l DNA Ligation Buffer and 10l DNA Ligation Mix at 20C for 15 mins, and purified with 0.8X DNA Purification beads with three 80% ethanol washes and elution with 15l Nuclease-free water. 10l of Universal dual indexes were ligated to the adapted cDNA by amplification with 25l KAPA HiFi HotStart ReadyMix (Roche Holding AG, Basel, Switzerland) at the following conditions of initialization at 98C for 45 secs, 12 cycles of denaturation at 98C for 15 secs, annealing at 60C for 30 secs, extension at 72C for 30 secs, a final extension at 72C for 1 min, and holding at 4C. The indexed cDNA library was purified with 1X DNA Purification beads with two 80% ethanol washes and elution with 20l Nuclease-free water. Purified indexed tick cDNA library was quantified with a Qubit dsDNA BR Assay kit (Invitrogen, Waltham, MA, USA) and library size profile visualized with a Bioanalyzer High Sensitivity DNA Assay kit (Agilent Technologies, Santa Clara, CA, USA).  500ng of the tick cDNA library was pooled in an 8-plex reaction (total 1,500ng), dried with a low heat vacuum concentrator, resuspended with 5l human Blocker solution and 7l Universal Blockers, and subsequently heated at 95C for 5 mins. Simultaneously, a 28l probe solution containing 20l Hybridization Mix, 4l Comprehensive Viral Research Panel and 4l custom probe panel was prepared, heated at 95C for 2 mins and immediately cooled on ice for 5 mins. Both cDNA library and probe solution were equilibrated to room temperature and combined, with the addition of 30l Hybridization Enhancer on the top of the mixture and incubated at 70C for 16 hrs. The hybridized reaction was added directly from the thermal cycler at 70C to 200l of Binding Buffer resuspended Streptavidin beads and incubated at room temperature, on a shaker for 30 mins. The enriched cDNA library bounded Streptavidin beads was washed once with 200l of Wash Buffer 1,  followed by three rounds of washing with 200l of 48C Wash Buffer 2, with 5 mins of incubation at 48C between each wash, and resuspended in 45l of Nuclease-free water. 22.5l of the Streptavidin Binding Bead slurry was amplified in a 50l reaction containing 2.5l ILMN Amplification Primers and 25l KAPA HiFi HotStart ReadyMix (Roche Holding AG, Basel, Switzerland) at the following conditions of initialization at 98C for 45 secs, 19 cycles of denaturation at 98C for 15 secs, annealing at 60C for 30 secs, extension at 72C for 30 secs, a final extension at 72C for 1 min, and holding at 4C. The amplified Streptavidin Binding Bead slurry was purified with 1.8X DNA Purification beads with two 80% ethanol washes and eluted with 30l Nuclease-free water. The eluted enriched library was quantified with a Qubit dsDNA BR Assay kit (Invitrogen, Waltham, MA, USA) and library size profile visualized with a Bioanalyzer High Sensitivity DNA Assay kit (Agilent Technologies, Santa Clara, CA, USA), followed by sequencing on an Illumina MiSeq, 150bp x 2 paired end reads.