Zika virus (ZIKV) strain PRVABC59 was used for all experiments. PRVABC59 was initially isolated in 2015 from a patient infected while in Puerto Rico. We obtained this strain from the Centers for Disease Control and Prevention in Fort Collins, CO. The virus used in these experiments has undergone a total of 5 passages in Vero cells. Viral titers were determined by plaque assay on Vero cells (ATCC®CCL-81™). ZIKV was UV-inactivated (UV-ZIKV) by exposing virus to UV light in a Spectroline UV Crosslinker for 1 hour. Vero cells were cultured in complete DMEM medium consisting of 1x DMEM (Corning Cellgro), 10% FBS, 25mM HEPES Buffer (Corning Cellgro), 2mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics, and were maintained at 37°C and 5% CO2.
Uv crosslinker
Manufactured by Spectroline
Sourced in United States
The UV Crosslinker is a laboratory equipment designed to expose samples to controlled doses of ultraviolet (UV) light. It is used to crosslink nucleic acids, proteins, and other biological molecules, enabling various applications in molecular biology, genetics, and biochemistry.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Atcc ccl 81, by American Type Culture Collection (1 mentions)
Hepes buffer, by Corning (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Sw55 rotor, by Beckman Coulter (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Streptavidin sepharose high performance bead, by GE Healthcare (1 mentions)
Pierce crosslink ip kit, by Thermo Fisher Scientific (1 mentions)
Ago2 antibody clone 11a9, by Merck Group (1 mentions)
Autoradiography film, by Kodak (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
9 protocols using uv crosslinker
1
Zika Virus Strain PRVABC59 Propagation
2
Probing mRNA Pathway in Initiation Complexes
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To examine the effect of DHX29 on the pathway taken by mRNA in the initiation complexes, we used a UV-cross-linking assay. The 48S ICs were reconstituted with/without DHX29 in an 80 µL reaction mixture with scaled amounts of the initiation components on co-transcriptionally 32P-labeled and 4-thioU-incorporated “+5,” “+7,” “+9,” “+11,” “+13,” and “+15” mRNAs. After assembly, the initiation complexes were purified from unbound components by centrifugation through 10%–30% SDG prepared in buffer A in a Beckman SW55 rotor at 53,000 rpm for 75 min, irradiated at 360 nm for 30 min on ice using a UV Crosslinker (Spectroline), and digested with 5 units RNase A for 10 min at 37°C. The samples were assayed by electrophoresis in NuPAGE 4%–12% Bis-Tris gel (Thermo Fisher Scientific) and autoradiography. After the RNase treatment of the samples, the cross-linked proteins acquired additional weight in the form of nucleotides, which resulted in a shifted band on SDS-PAGE. For the UV-cross-linking experiment with the eIF3/mRNA binary complex, we repeated this protocol but omitted the SDG purification step.
3
UV-Induced S. acidocaldarius Cell Aggregation
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UV light exposure of S. acidocaldarius cells was performed as described previously [30 (link)]. Ten millilitres of culture (OD600 0.2–0.3) was induced with 75 J m−² UV light (254 nm, Spectroline UV-crosslinker) in a Petri dish. After that, cultures were incubated at 75 °C for 3 h. Subsequently, 5 ml of each culture (diluted to OD600 0.2) was spotted on a microscope slide coated with 1 % agarose. Single and aggregated cells (n> 3) were analysed by an ImageJ cell counter (NIH, Bethesda, MD) from at least three independent experiments. The percentage of cells found in aggregates was subsequently calculated.
4
Examining DHX29 Effect on Initiation Complexes
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To examine the effect of DHX29 on the rearrangement of initiation complexes, we performed UV crosslinking experiments. The 48S ICs were reconstituted with/without DHX29 and eIF1A on co-transcriptionally 32P-labeled ‘−3U’ and ‘+4U’ mRNAs containing 4-thioU at the −3 and +4 key context positions, respectively. To maintain the integrity of ribosomal complexes, we omitted the purification step by SDG centrifugation. After assembly, the 48S ICs were irradiated at 360 nm for 30 min on ice using a UV Crosslinker (Spectroline) and were then digested with 5 units RNase A for 10 min at 37°C. To identify crosslinked proteins, ribosomal complexes were assayed by SDS-PAGE and autoradiography.
5
Synthesis and Analysis of Biotinylated RNA
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For synthesis of biotin-labeled RNA transcript, the EV-A71 genomic regions, 5′-UTR and 3′-UTR were constructed in pZback/blunt linearized vector (BioTools Co., Ltd). The blunt-end PCR products generated by proofreading DNA polymerases can be directly ligated with the pZBack/blunt vector, followed by the in vitro transcription. RNA transcripts were synthesized using the MEGAscript T7 kit (Thermo Fisher Scientific Co., Ltd, Waltham, MA, USA) according to the manufacturer’s protocol. In brief, the biotinylated RNA was produced in an 20 μL MEGAscript transcription reaction with 20 mM biotinylated UTP. The synthesized RNAs were then purified and analyzed on 1% agarose gel. To carry out the co-immunoprecipitation, 8 μg of biotinylated RNAs were added into Streptavidin Sepharose High Performance beads (GE Healthcare), following by adding the recombinant MBP-Stau2 protein and incubation for 4 h. This reaction received the UV cross-link in the UV CROSSLINKER (Spectroline, Westbury, NY, USA) to enhance the binding of RNA and recombinant protein. The bound eluates were then analyzed by western blotting.
6
Identification of miR-106b targets using Ago2-IP
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BxPc-3 cells were mock transfected or transfected with biotinylated has-miR-106b mimic. Forty-eight hours post-transfection, cells were placed on ice, irradiated once with 150 mJ/cm2 at 254 nm using a UV Crosslinker (Spectroline, Westbury, NY) and lysed for 15 minutes on ice in lysis buffer. Five mg of of post-nuclear extracts were subjected to immunoprecipitation using 10 μg of Ago2 antibody (clone 11A9, EMD Millipore, Billerica, MA, USA) or 10 μg of rat IgG using Pierce Crosslink IP Kit (Thermo Scientific, Pittsburgh, PA). The rest of the immunoprecipitated complex was digested with 30 μg of proteinase K to release the ribonucleoprotein complex and a second round of immunoprecipitation was performed using anti-streptavidin antibody (EMD Millipore). RNA extraction and qRT-PCR was done as described above. Relative enrichment was calculated from the qRT-PCR data.
7
UV Treatment Impacts on S. acidocaldarius Survival
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UV treatment of S. acidocaldarius was performed as described by Wolferen et al. (2015) (link). To determine cell survival rates, 10 mL culture (OD600 0.2–0.3) was poured in plastic petri dishes and treated with different doses of UV light (75, 125, and 200 J/m2) (254 nm; Spectroline UV cross-linker). A culture without UV treatment was used as control. All the cultures were serially diluted in medium and spread on solid gelrite plates. Plates were cultivated at 75°C for 3–4 days until colonies were visible. Colonies from UV treated and non-UV treated samples were counted. To quantify the cell survival rates, data from at least three independent experiments was used. For analyzing ups pili dependent cell aggregation, a dose of 75 J/m2 UV light was used. Samples without UV treatment were used as the control. After UV treatment, samples were cultivated at 75°C for 3 h and analyzed with phase-contrast microscopy.
8
Propagation and Inactivation of HSV-2 Viruses
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Vero and U2OS cells were obtained from the American Type Culture Collection (Manassas, VA). Vero and U2OS cells were propagated in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 mg/ml streptomycin, hereafter referred to as “complete DMEM.” Wild-type HSV-2 MS (ATCC) and HSV-2 MS-GFP [15 (link)] were propagated and titered on Vero cells. The HSV-2 ICP0- mutant virus, HSV-2 0ΔNLS [15 (link)], was propagated and titered in U2OS cells. Ultraviolet (UV) inactivation of HSV-2 0ΔNLS was achieved by placing 1.5 ml of virus solution in a 60-mm dish in a Spectroline UV crosslinker, and delivering four successive cycles of 1 mJ/cm2 of radiation (i.e., instrument set to '9999' x 100 μJ/cm2) to achieve a total dose of 4 mJ/cm2.
9
Quantifying m6A RNA Methylation
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