Pes syringe filter

Cells were detached using Accutase (Sigma–Aldrich) and washed 2x with ice-cold phosphate-buffered saline (PBS, Sigma–Aldrich). Cells were lysed in 1x TBS (pH 7.4) containing 0.5% NP40 and a protease/phosphatase inhibitor cocktail (Pierce, #A32959). Then the lysates were filtered through a 0.22 µm PES syringe filter (Millipore). About 10 mg lysates were immunoprecipitated with prewashed M2 anti-flag agarose resin (Sigma–Aldrich, #A2220) for 2 h at 4 °C while rotating. For Flag coimmunoprecipitation, Flag was eluted with Flag Peptide (Sigma–Aldrich, #F3290) by moving on a wheel for 20 min at 4 °C followed by a spin for 1 min at 1000 × g. The flow-through was collected and subjected to TMT labeling or Western blot analysis. The antibodies are listed in Supplementary Table 3.

sgRNA-expressing and lentiviral packaging plasmids (VSVg/Delta8.9) were transiently co-transfected into 293T cells with Lipofectamine 2000. Lentiviral supernatants were harvested at 72 h and filtered through a 0.45 µm PES syringe filter (Millipore). Transduction with lentivirally encoded sgRNAs performed as described in Supplementary Data 1 with cell line-specific protocols. 3 days following lentiviral infection, cells were started on Zeocin selection at cell line-specific concentrations (Supplementary Data 1) in order to select for sgRNA-expressing cells. Prior to gene expression analysis, uniform selection of sgRNA-infected populations was confirmed by flow cytometric analysis of the co-expressed mTagBFP2 reporter.

Swabs from the infected patients were collected in 400 µl 1XPBS buffer containing penicillin (20 IU/ml), streptomycin (100 µg/ml) and amphotericin B (250 µg/ml). They were filtered using 0.22 µm PES syringe filter (Millipore, Ireland). Approximately, 200 µl of the filtrate was mixed with DMEM1 to make the volume up to 1 ml before inoculating monolayer of A549 or Vero cells in T25 flasks. The culture flask was incubated for 1 h at 37 °C in a CO₂ incubator for virus adsorption. This was followed by topping the cell monolayer with 4 ml of DMEM (1% FBS).
Plaque forming viruses were titrated and plaques were picked at 72 h (P.I.). Plaques from each cytopathic isolate were subjected to three cycles of plaque-purification and labelled as 3PP (i.e., HM3PP, PB3PP etc.). Stocks were prepared for each isolate for downstream applications.

Virus produced from HEK 293T cells was centrifuged to remove cells, filtered through a 0.45 μm Polyethersulfone (PES) syringe filter (Millipore, Burlington, Massachusetts), transferred into 25 × 89 mm polyallomer ultracentrifuge tubes (Beckman Coulter, Brea, California), and ultracentrifuged in an Optima XL-100K ultracentrifuge (Beckman Coulter) using an SW28 rotor at 25,500 rpm (117,250 × g) for 2.5 h at 4°C. Following aspiration of supernatant, the pellet was fixed in FGP fixative (1.25% formaldehyde, 2.5% glutaraldehyde, and 0.03% picric acid in 0.1 M sodium cacodylate buffer (pH 7.4)) for 2 h at room temperature and stored at 4°C. Ultrathin sections (60 nm) were cut on a Reichert Ultracut-S microtome, transferred to copper grids stained with lead citrate, and observed using a JEOL 1200EX microscope with an AMT 2k charge-coupled-device camera. Images captured at 30,000× magnification were visually inspected to classify viral particles as mature, immature, eccentric, or containing 2 apparent capsids, and over 100 particles were counted per virus preparation; representative TEM images are shown in S20 and S21 Figs and summarized in S8 Table. Statistical significance versus matched WT morphology was assessed using 2-way ANOVA with Sidak multiple comparisons test with GraphPad Prism software.

Chicken juice was prepared as described previously (Brown et al., 2013 (link), 2014 (link)). Briefly, frozen commercially available whole chickens were purchased from UK supermarkets before thawing at room temperature. Exudate was collected, centrifuged to remove debris and sterilized by using a 0.2 μm sterile polyethersulfone (PES) syringe filter (Millipore) before aliquotting and storage at −20°C until use. Chicken juice was diluted v/v in Brucella medium for use in biofilm assays.

DAPT treatment was carried out as previously described11 (link). In brief, 50 mM DAPT stock solution (Merck) was prepared in 100% DMSO (Sigma) and stored in a small volume at −20 °C. Embryos in their chorions were transferred to 12-well plates at 2 h.p.f. in 1.6 ml E3 medium with 20 embryos per well. A total of 50 μM DAPT in E3 medium was prepared immediately before the treatment. To prevent precipitation, the DAPT stock solution was added into E3 medium while vortexing, and then filtered by 0.22 μm PES syringe filter (Millipore). DAPT treatment was initiated at dome stage (4 h.p.f.) by replacing E3 medium with E3/DAPT medium. For pulse-chase experiments, DAPT was washed out at least twice with fresh E3 medium +0.03% PTU. Embryos were dechorionated and fixed at 36 h.p.f. All experiments were incubated at 28.5 °C, except for short operations, for example, washing out, which were at room temperature.