CellTag barcoding plasmids containing constitutively expressed GFP construct were packaged into lentivirus with a titer of 2.5–3.5 × 108 TU/mL [15 (link),16 (link)]. A 200 μL aliquot of lentiviral Library #1 was added to low-passage naive RB028 cells in a 24-well plate, and the culture was centrifuged for 1 h at 1000× g in 32 °C. Cells were allowed to recover for at least one week. After a GFP signal was observed, cells were manually dissociated over ice with a P1000 pipette (pipetting 30–40 times) and passed through a 30 μm cell strainer. GFP+ RB028 cells were selected using fluorescence-activated cell sorting (FACS) with BD FACS SORP Aria-IIu, reaggregated using anti-adherence-rinsing-solution-treated microwell plates (STEMCELL Technologies, catalogue #34411 and #07010) and recovered overnight with rock inhibitor Y-27632 (10 μM; Selleckchem, catalogue #S1049). Cell clusters were lifted one week later and transferred into a flask for routine cell culturing protocol and then mechanically dissociated for each of the scRNA-seq experiments. In total, 10,000 cells were targeted for capture using 10X Illumina 3v3.1′ chemistry. The remaining cells were reaggregated into microwell plates with Y-27632 overnight. Once the culture recovered, cells were treated with 1 μM carboplatin to generate chemoresistance as described above.
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