Orca 4.0 camera

FISH was performed by the St. Jude Cytogenetics core facility. Lin⁻ cells were incubated with colcemid for 4 h then harvested by routine cytogenetic methods. For FISH analysis, BAC clones were purchased from BACPAC Resources (bacpacresources.org; Children's Hospital Oakland Research Institute, Oakland, CA, USA), labeled with either red-dUTP (AF594, Molecular Probes) or green-dUTP (AF488, Molecular Probes), and used as hybridization probes in signal segregation studies (Table S3). Labeled probe pairs were combined with 100 ng/ml sheared mouse DNA and hybridized to interphase and metaphase cells in 50% formamide, 10% dextran sulfate and 2× saline sodium citrate (SSC) at 37°C for 16 h. Cell nuclei were stained with 2.5 mg/ml 4′,6-diamidino-2-phenylindole (DAPI), imaged using a Nikon E800 microscope (Nikon PlanApo 60×/1.40 NA oil objective), Nikon Nis Elements software and a Hamamatsu Orca 4.0 camera.

Cells were moved from agar plates into liquid 384‐well plates using the RoToR bench‐top colony arrayer (Singer Instruments). Liquid cultures were grown overnight in synthetic medium with 2% glucose (SD) in a shaking incubator (LiCONiC Instruments) at 30°C. A Tecan freedom EVO liquid handler (Tecan), which is connected to the incubator, was used to back‐dilute the strains to ∼ 0.25 OD₆₀₀ in plates containing the same medium. Plates were then transferred back to the incubator and were allowed to grow for 4 h at 30°C to reach logarithmic growth phase. The liquid handler was then used to transfer strains into glass‐bottom 384‐well microscope plates (Brooks Bioscience) coated with Concanavalin A (Sigma‐Aldrich) to allow cell adhesion. Wells were washed twice in a low fluorescence synthetic medium (Formedium) to remove floating cells and reach a cell monolayer. Plates were then transferred into the automated microscopy system using a KiNEDx robotic arm (Peak Robotics).
Imaging was performed using an automated Olympus SpinSR system using a Hamamatsu flash Orca 4.0 camera and a CSUW1‐T2SSR SD Yokogawa spinning disk unit with a 50 μm pinhole disk. Images were acquired using a 60× air lens NA 0.9 (Olympus), 100 mW 488 nm OBIS LX laser system (Coherent), GFP Filter set [EX470/40, EM525/50] (Chroma).
Images were manually inspected using Fiji‐ImageJ software (Schindelin et al, 2012 (link)).

Recombinant GFP tagged RSV A2 strain was kindly provided by Fix et al.^{23 (link)}. Viral propagation was performed in HEp-2 cells (MOI 0.1) for 3–5 days in Opti-MEM with 1mM L-glut and 2% NCS. Infected cells were lysed in a sonicating water bath (Grant, XUBA1), followed by centrifugation at 1200 rpm for 5 minutes. Supernatant was collected and virus concentrated and purified as described previously^{24 (link)}. Briefly, virus was purified by centrifugation through a polyethersulphone membrane with a pore size of 1000 000 Daltons MWCO (1000 kD) (Vivaspin-20, Vivascience, Gloucester, UK). Virus was collected in RPMI (Life Technologies), aliquoted and frozen at −80 °C. Viruses were quantified by plaque assay. Briefly, 2.5 × 10⁴ HEp-2 cells were seeded into a 96 well plate and cultured overnight in Opti-MEM plus supplements (as above) at 37 °C 5% CO₂. The following day cells were washed in PBS and serial tenfold dilutions of viral stock added to triplicate wells (50 µl) for 2 hours at 37 °C 5% CO₂. The inoculum was replaced with 200 µl of Opti-MEM for 24–48 hours at 37 °C and 5% CO₂. Images of the whole well were captured using a Nikon Eclipse Ti-U microscope equipped with Hamamatsu ORCA 4.0 camera and fluorescence FITC filter. The number of viral plaques from each well were counted and pfu/ml was calculated by multiplying the plaques/well by dilution factor and x10.