Sh800

The mutant library was transduced into 293T cells via spinfection. To find optimal virus volumes for achieving a multiplicity of infection (MOI) of 0.1–0.3, each library virus was tested by spinfecting 5×10⁵ cells with several different volumes of virus in the 6-well plate. Each well received a different titrated virus amount (usually between 50 and 500 μl) along with a no-transduction control and infected cell rate was determined by anti-HA Alexa 594. Then, four 6-well plates, 1.2 ×10⁷ cells were centrifuged at 1000 g for 1.5 h at 37 °C.
Cells were sorted using a SH800 (SONY) 24 h after spinfection. Approximately 5×10⁷ induced library cells were resuspended with complete medium and incubated for 30 min at 4 °C with a 1/40~1/160 dilution of medium containing RBD-sfGFP and a 1/4000 dilution of anti-HA Alexa 647 (clone TANA2, MBL). Cells were directly sorted on SH800 (SONY). The top 0.05% of cells were sorted and their genomic DNA was extracted by NucleoSpin Tissue (TAKARA). Mutated ACE2 fragment was cloned into pcDNA4TO ACE2 for individual validation and also introduced random mutations again with error-prone PCR for further screening.

sgRNAs for CRISPRi of KSHV (accession number GQ994935.1) were designed as described by Horlbeck et al. [33 (link)] (Table S1). Synthetic DNA segments encoding the sgRNAs were cloned into the pHR-SFFV-dCas9-BFP-KRAB (Addgene 46911) at BstXI and XhoI, and the clones were confirmed by sanger sequencing. Lentiviral production and transduction were performed as described above. After transduction, BCBL-1 cells were maintained in 1 μg/mL of puromycin for 10 days prior to the selection of BFP+/sgRNA+ by FACS in a Sony SH800 instrument. iSLK-219 cells were selected for BFP+/sgRNA+ expression by FACS in a Sony SH800 instrument (see Tables S1 and S2 for sgRNA sequences and genomic coordinates).

The cell images were captured using a microscopy BZ-X700 (Keyence). The relative cell size was measured by flow cytometry using a SH800 cell sorter (SONY). The cells were harvested at 48 h and tenfold diluted sample with PBS was analyzed. The flow rate was set to 6 μL/min (pressure 1). Forward scatter data and back scatter data of 100,000 counts was recorded using SH800 software (SONY).

Fresh human PBMCs were stained with monoclonal antibodies, such as PercP-Cy5.5-anti-human CD3, APC/Cy7-anti-human CD4, PE-anti-human CD25, and Brilliant Violet 510-anti-human CD127 (all purchased from BioLegend, San Diego, CA, USA), in 0.5% BSA + 2 mM EDTA in PBS. Splenocytes were obtained from the spleens of mice by mechanical disruption and through a 200-gauge steel mesh. Splenocytes were lysed with 1_X red blood cells (RBCs) lysis buffer to remove RBCs. First, magnetic microbeads (Miltenyi Biotec) bead-based enrichment of CD4⁺T cells was performed. Then, cells were stained with monoclonal antibodies, such as PE-anti-mice CD4, APC/Cyanine7-anti-mice CD25, and Alexa-Fluor647-anti-mice CCR6 (all purchased from BioLegend), in 0.5% BSA + 2 mM EDTA in PBS. Human Treg cells and murine Treg cells were sorted using a SH800S cell sorter (SONY, Japan) and analyzed with the SH800 software (SONY, Japan). Human Treg cells were gated as CD3⁺CD4⁺CD25⁺CD127^- within the lymphocyte gate, which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). Mice Treg cells were gated as CD4⁺CD25⁺ within the lymphocyte gate which excluded cell debris, doublets, and dead cells (Supplementary Figure 2). The purity of the sorted cell population was above 95% for human and 91% for mice, which was verified using post-sorting flow cytometry.

The volumetric cell density (cells/l) was measured by flow cytometry using a SH800 cell sorter (SONY). Cells grown under aforementioned conditions were harvested at 48 h (OD₆₀₀ between 20 and 25) and 10-fold diluted sample with water was analyzed. The flow rate was set to 11 μl/min (pressure 2). All FSC (forward scatter) and SSC (side scatter) images were recorded using SH800 software (SONY).