Ez link psoralen peg3 biotin

Infection experiments were performed under biosafety level 3 conditions. SARS-CoV-2 virus strain Wuhan-Hu-1 was kindly provided by (Wuhan Institute of Virology). Independent biological replicates were performed using 90–120 million cells each. VeroE6 cells were inoculated with SARS-CoV-2 strain Wuhan-Hu-1 at a multiplicity of infection = 0.01 pfu/cell for 24 h. Following inoculation, two flasks of cells were washed three times by phosphate-buffered saline (PBS) and then subjected to crosslinking. The remaining cells were cultured for another 48 h, when CPE was observed in about 70% cells, supernatant was collected and centrifuged at 4 °C 200 × g for 10 min to remove cell pellet. Then the clear supernatant was mixed with equal volume of saturated ammonium sulfate and incubate at 4 °C for 1 h. At the same time, the remaining unshed cells were washed three times by PBS and subjected to crosslinking.
For crosslinking, cells or virus pellet were incubated with 2 mM of EZ-Link Psoralen-PEG3- Biotin (ThermoFisher Scientific) at 37 °C for 10 min in PBS containing 0.01% digitonin. The cells were then spread onto a 10 cm plate and irradiated using 365 nm ultraviolet radiation for 20 min on ice. Cell and virion RNAs were extracted with RNeasy mini kit (Qiagen).
The virus inoculation and crosslinking were performed on three independent replicates.

T1L viral dsRNA was extracted from purified T1L virions by TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher # 10296010). Viral dsRNA was conjugated with EZ-Link Psoralen-PEG3-Biotin (Thermo Fisher # 29986) by UV irradiation (365 nm) on ice for 30 min as described [99 (link)]. Excess biotin was removed by precipitation of the biotinylated-dsRNA using 0.3 M potassium acetate and 2.5 volume of ethanol. Biotinylated-dsRNA was added to cell lysates expressing various proteins and incubated at 4°C for 2 h. Pierce Streptavidin Agarose (Thermo Fisher # 20353) was added into the mixtures and incubated at 4°C for another 2 h. Agarose was washed five times with lysis buffer and immunoblotted to detect the dsRNA-binding capacity of the various proteins.

The intercalation of bio-psoralen into underwound DNA was examined using an in vitro crosslinking assay^{26 (link)}. DNA was crosslinked by bio-psoralen under UV irradiation, linearized, and denatured by heat. As bio-psoralen crosslinking prevents DNA heat denaturation, the intercalation of bio-psoralen can be evaluated based on the amount of non-denatured DNA after heat treatment. Relaxed and underwound DNA were prepared by incubating plasmid pBR322 (Takara Bio) with topoisomerase I (TopI, Takara Bio) and gyrase (TopoGEN), respectively, at 37 °C for 1 h, and purified using NucleoSpin^® Gel and a PCR Clean-up kit (Macherey-Nagel). Bio-psoralen (200 µM; EZ-Link^® Psoralen-PEG₃-Biotin, Thermo Fisher Scientific) was added to DNA and exposed to 3.6 kJ/m² of long-wave (365 nm) UV (LUV-4; AS ONE) on ice for 30 min. For linearization, DNA was cleaved with EcoRI (Toyobo) at 37 °C for 1 h. For heat denaturation, one half of each sample was boiled for 5 min and immediately chilled in ice-cold water. Agarose gel electrophoresis was run at 50 V for 1 h, and the gel was stained with 0.1 µg/mL ethidium bromide solution (Nacalai Tesque) for 30 min. Images were acquired using ImageQuant^TM LAS500 (Cytiva) and DNA bands were quantified using Fiji^{59 (link)}. The fraction of bio-psoralen-crosslinked DNA was calculated by dividing the amount of non-denatured DNA by the total amount of DNA.