gRNAs were produced in vitro using previously published methods42 (link). Briefly, overlapping oligomers containing a T7 promoter, the desired protospacer, and gRNA scaffold were amplified using Phusion polymerase (New England Biolabs). The unpurified DNA product was then subjected to in vitro transcription using the NEB HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs), incubating at 37 °C for 16 h. The following day, RNA was treated first with DNase I followed by rSAP (recombinant Shrimp Alkaline Phosphatase; New England Biolabs), purified with the miRNeasy kit (Qiagen), concentration measured by Nanodrop, and frozen at −80 °C.
Neb hiscribe t7 high yield rna synthesis kit
Manufactured by New England Biolabs
The NEB HiScribe T7 High Yield RNA Synthesis Kit is a tool for in vitro transcription of RNA. It utilizes the T7 RNA polymerase to generate high yields of RNA from DNA templates.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy kit, by Qiagen (1 mentions)
Phusion polymerase, by New England Biolabs (1 mentions)
Agpath id one step rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Am8740, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra 2 non directional rna second strand synthesis module, by New England Biolabs (1 mentions)
4 protocols using neb hiscribe t7 high yield rna synthesis kit
1
In Vitro gRNA Production Protocol
2
Duplex RT-PCR for Hendra Virus Variant Detection
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We adapted quantitative RT-PCR targeting the HeV M gene (28 (link)) to target HeV-var. The duplex assay used the Applied Biosystems AgPath-ID One-Step RT-PCR kit (ThermoFisher) and distinguishes prototype and variant HeV strains. In brief, we combined 4 μL RNA with 10 μL 2× RT-PCR buffer, 0.8 μL 25× RT-PCR enzyme mix, 2 μL nuclease-free water, and 3.2 μL primer/probe mix (0.6 μL each primer, 0.3 μL each probe from 10 μmol stock; Table 1 ). We generated the reaction using 10 min at 50°C for cDNA synthesis, 10 min at 95°C for RT inactivation, and 50 cycles of 95°C for 15 s and 60°C for 30 s with FAM and HEX channels captured at the end of each cycle. As positive control, we synthesized gene fragments encoding a T7 promoter upstream of the partial M gene for both prototypic and variant HeV (Appendix Figure 1). We expressed RNA transcripts using the NEB HiScribe T7 High Yield RNA Synthesis kit (New England Biolabs, https://www.neb.com ).
3
RNA Amplification and Fragmentation Protocol
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Following this, we performed second strand synthesis using the NEBNext Ultra II Non-Directional RNA second strand synthesis module as per the suggested protocol (NEB, Catalog# E6111L). The synthesized DNA was purified via 1X Mag-Bind TotalPure NGS beads and eluted in ~12 ul of sterile water. 10 ul of this was then used as an input for T7 polymerase mediated In vitro Transcription (IVT) using the NEB HiScribe T7 High Yield RNA Synthesis Kit (NEB, # E2040S). Briefly, all the components were mixed as mentioned in the kit protocol and incubated at 37°C (lid at 50°C) for 16 hours. The reaction was eluted in 20 μl of sterile water after a round of 1X Mag-Bind TotalPure NGS bead cleanup. This newly transcribed RNA was quantified using a Nanodrop and to improve the hybridization kinetics and enhance signal, 500ng of the amplified RNA was fragmented by RNA Fragmentation reagent in a total reaction volume of 10 μl as per specifications (Thermo Fisher, Catalog# AM8740).
4
Efficient RNAi Knockdown in S2 Cells
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S2 cells were cultured in M3+BPYE medium, made as directed from Shields and Sang Powdered Medium (Sigma S-8398), supplemented with 0.5 g KHCO3, 1 g yeast extract and 2.5 g bactopeptone per liter, pH adjusted to 6.6 and sterile-filtered. 100x Antibiotic-Antimycotic (Thermo-Fisher 15240062) and Fetal Bovine Serum (Sigma F2442 Lot # 078K8405) were added to concentrations of 1x and 10%, respectively. Cells were maintained at 25°C and passaged every 3-4 days for 7 passages prior to use for RNAi experiments.
For dsRNA-induced knockdown, cells were plated in serum-free medium at a concentration of 2.5 million cells/ml, then treated with 30 µg/ml of lacZor stwl-dsRNA for 60 minutes before addition of M3/BPYE medium containing 13% FBS (final concentration, 10% FBS, 7.5 µg/µl dsRNA). Cells were chemically cross-linked and frozen after 3 days. RNA was synthesized using NEB HiScribe™ T7 High Yield RNA Synthesis Kit (E2040S) from PCR products generated from YEp365 plasmid (lacZ control) or genomic DNA extracted from S2 cells. For efficient stwl KD we generated three distinct dsRNAs from reference, each targeting the second exon of stwl, which is present in all stwl transcripts (Hu et al., 2016) . S2 cells were treated with 10 µg/ml of each dsRNA.
For dsRNA-induced knockdown, cells were plated in serum-free medium at a concentration of 2.5 million cells/ml, then treated with 30 µg/ml of lacZor stwl-dsRNA for 60 minutes before addition of M3/BPYE medium containing 13% FBS (final concentration, 10% FBS, 7.5 µg/µl dsRNA). Cells were chemically cross-linked and frozen after 3 days. RNA was synthesized using NEB HiScribe™ T7 High Yield RNA Synthesis Kit (E2040S) from PCR products generated from YEp365 plasmid (lacZ control) or genomic DNA extracted from S2 cells. For efficient stwl KD we generated three distinct dsRNAs from reference, each targeting the second exon of stwl, which is present in all stwl transcripts (Hu et al., 2016) . S2 cells were treated with 10 µg/ml of each dsRNA.
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