Guava easycyte instrument

A previously described LMN shRNA library targeting chromatin regulatory proteins was used (Zuber et al., 2011b (link)). Additional shRNAs were included in the library to allow comprehensive targeting of all bromodomain-containing proteins. shRNA plasmids from this library were arrayed individually in 96 well plates at 50 ng/µl for retrovirus preparation using Ecotropic Plat-E cells as described (Morita et al., 2000 (link)). Viral transduction of B-ALL cells was also performed in 96 well plates, followed by flow-cytometry-based measurement of GFP positivity using a Guava Easycyte instrument (Millipore, Billerica, MA). Measurements were performed on day 2 and day 12 post-transduction. Renilla and Rpa3 control shRNAs were included with each 96 well plate as negative and positive controls, respectively. If an shRNA was introduced into B-ALL cells with a day 2 GFP percentage below 5%, this sample was discarded from the screen. The lower limit on GFP% measurements on day 12 was arbitrarily set at 0.2%. A single instance of Renilla and Rpa3 control shRNAs are included in the screen histogram shown in Figure 1A. The screen data can be found in Supplementary file 1.

293T cell suspensions were transduced via spinoculation for 1 h at 500 × g at 4°C in the presence of 8 μg/mL Polybrene. The percentage of RFP-positive cells is indicative of the transduction efficiency and was used as an indicator of ISG expression levels. Forty-eight hours postransduction, the cell populations were split and infected with 50 μL of BTV-8-mGFP virus for a period of 16 h or 32 h. The dose of BTV-8-mGFP had previously been optimized to ensure that 10 to 50% of cells in each well were infected. For the late screen, a final concentration of 0.625 μg/mL of puromycin was used to supplement the medium, enabling virus replication to be limited to the transduced cells. At 16 and 32 hpi, cells were washed, trypsinized, and fixed in 4% (wt/vol) formaldehyde in PBS. Cells were then analyzed by flow cytometry, using the Guava easyCyte instrument (Millipore) equipped with a 488-nm and a 532-nm laser. For each well, the single live cell population was gated and levels of GFP and RFP were measured. ISGs with transduction levels (measured by RFP expression) below 10% were excluded from downstream analyses. Data were then analyzed using FlowJo (TreeStar) software.