Fitc anti mouse lineage cocktail

Tibias and femurs were collected from eight mice (four non-tumor bearing and four glioma bearing), and lineage⁻ HSPCs were obtained using the same protocol as previously. To obtain a pure population of lineage⁻ HSPCs, cells were stained with LIVE/DEAD Fixable Violet (ThermoFisher cat. L34963) and FITC anti-mouse Lineage Cocktail (Biolegend cat. 133302) and sorted on a Sony SH800 Cell Sorter. Lin⁻ purity of over 95% was achieved for all samples. The bone marrow derived cells were measured directly after sorting, and a cell suspension volume equivalent to 10,000 target cells was used for further processing. Cells were diluted in ice-cold phosphate-buffered saline (PBS) containing 0.4% bovine serum albumin (BSA) at a density of 1,200 cells/µL. Cells were loaded into a Chromium NextGEM Chip G (10× Genomics, Pleasanton, California, USA) and processed in Chromium X following the manufacturer’s instructions. Preparation of gel beads in emulsion and libraries were performed with Chromium Next GEM Single Cell 3′ Kit V.3.1 (Dual Index) according to user guide provided by the manufacturer. Libraries quality and quantity were verified with 2200 TapeStation (Agilent Technologies, USA). Libraries were pooled based on their molar concentrations. Pooled library was sequencing on one high-output lane of the NovaSeq 6000 instrument (Illumina, San Diego, California, USA).