title: Single-cell transcriptional profiling of healthy brain, primary and post-irradiation recurrent murine glioblastomas using 10x Genomics Flex chemistry
library_construction_protocol: Fresh tumors and healthy brain tissue were dissected from Nestin-Tv-a RCAS mice. Tissue was immediately processed into single-cell suspensions using the Adult Brain Dissociation Kit (Miltenyi Biotec, 130-107-677) according to the manufacturer's instructions. Cell viability was assessed by trypan blue exclusion, and samples with viability above 70% were carried forward for fixation. No nucleic acid extraction was performed prior to library preparation. Instead, single-cell suspensions were fixed using the Chromium Fixed RNA Sample Preparation workflow (10x Genomics). Cells were incubated in freshly prepared 4% formaldehyde diluted in Conc. Fix & Perm Buffer (10x Genomics, PN-2000517) for 24 h at 4 °C. Fixation was quenched according to protocol CG000478, and samples were stored in 50% glycerol at –80 °C until Flex library preparation. Whole-transcriptome libraries were generated using the Chromium Next GEM Single Cell Fixed RNA Flex Kit (10x Genomics, 1000568). Fixed cells were hybridized with the Chromium Mouse Transcriptome Probe Set v1.0.1 (10x Genomics) following protocol CG000478. Cells were partitioned into GEMs on a Chromium Controller, and libraries were constructed according to the manufacturer's instructions.  Five Flex hybridization reactions were performed. Across these hybridizations, a total of 23 individual biological samples were processed, including one thechnical replicate. Two samples exhibited near-zero UMI counts and were excluded after Cell Ranger processing. An additional five samples failed downstream QC, resulting in 16 samples retained in the final processed dataset.  Custom Flex probes targeting RCAS-derived transgenes (hPDGFB-HA, RFP, shP53, Tva; GFP as negative control) were evaluated in the first two pilot experiments (v1 and v2). One hPDGFB probe and two RFP probes showing robust performance were included in the final custom addon panel (v3). Custom probe sequences are provided in the supplementary material. 