title: scRNA-seq of BT142 cells co-cultured with non-mGAMs
design: BT142, a grade III IDH1-mutant anaplastic astrocytoma cell line 45, was purchased from ATCC. BT142 cells and previously sorted GAMs were centrifuged at 300g for 5 mins, washed with PBS and centrifuged again. The supernatants were discarded, and the pellets were resuspended at a concentration of 100 cells/uL using BT142 growth media consisting of DMEM/F12 basal media (Gibco, USA) supplemented with B27 without Vitamin A (Gibco), EGF (20 ng/mL; R&D Systems, USA), FGF2 (20 ng/mL; R&D Systems, USA), heparan sulfate (2 mg/mL; R&D Systems), and PDGF-AA (100 ng/mL; Peprotech, USA). BT142 cells were then mixed with mGAMs or non-mGAMs at a 1:1 ratio. Mixed cells were seeded into wells of an ultra-low attachment 96-well U-bottom plate (PrimeSurface; S-Bio, Japan) at a concentration of 10,000 cells per well in a final volume of 100uL. Wells seeded with only 5,000 cells of BT142 were also prepared. The plate was then centrifuged at 90g for 2 min. Cells were cultured in a humidified 37C, 5% CO2 incubator, with partial media changes every 2 days. Cells were observed daily under an inverted bright-field microscope for spheroid formation. At day 11, spheroids were dissociated using the Papain dissociation kit (Worthington Biochemical, USA) for downstream analyses. For Single-cell RNA-seq, cells were stained for CD45 (BioLegend, USA) and the CD45-negative cells (BT142) were sorted through FACS.  The BT142 cells were sequenced, and the data was processed.