biostudies-arrayexpressDavid LindgrenMus musculusCell Ranger (v7.1.0, cellranger count)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16532This study characterizes the single-cell transcriptional landscape of murine glioblastoma generated using the RCAS/Nestin-Tv-a system, in which hPDGFB and shTP53 transgenes drive tumor initiation. We profiled primary and post-irradiation recurrent tumors, along with healthy brain controls, using the 10x Genomics Flex (GEM-X) gene expression platform and custom probe addon sets targeting the RCAS transcripts. A total of 16 samples were included after QC filtering (primary tumors, recurrent tumors, and control brains). The data set provides genome-wide single-cell RNA-seq measurements at probe-level resolution.biostudies-arrayexpressSequencing - Flex libraries were sequenced on an Illumina NovaSeq 6000 (200 cycles). Sequencing was performed in paired-end mode. Base calling and demultiplexing were performed using the standard NovaSeq control software.Library Construction - 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.Sample Collection - 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.Nucleic Acid Extraction - 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.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw FASTQ files generated from 10x Genomics Single Cell Flex libraries were processed using Cell Ranger (v7.1.0, cellranger count). A custom mouse reference was generated with cellranger mkref, based on the mm10 (refdata-gex-mm10-2020-A) genome, Mouse Transcriptome Probe Set v1.0.1, and extended with additional transgene sequences (human PDGFB, RFP, and TVA) to enable detection of RCAS/Tv-a–derived transcripts. Processed output from the cellranger analysis is provided as `_sample_filtered_feature_bc_matrix.h5` files.Sequence Alignment - Raw FASTQ files generated from 10x Genomics Single Cell Flex libraries were processed using Cell Ranger (v7.1.0, cellranger count). A custom mouse reference was generated with cellranger mkref, based on the mm10 (refdata-gex-mm10-2020-A) genome, Mouse Transcriptome Probe Set v1.0.1, and extended with additional transgene sequences (human PDGFB, RFP, and TVA) to enable detection of RCAS/Tv-a–derived transcripts. Processed output from the cellranger analysis is provided as `_sample_filtered_feature_bc_matrix.h5` files.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAMus musculusAlexander PietrasDavid LindgrenfalseSingle-cell transcriptional profiling of healthy brain, primary and post-irradiation recurrent murine glioblastomas using 10x Genomics Flex chemistryThis study characterizes the single-cell transcriptional landscape of murine glioblastoma generated using the RCAS/Nestin-Tv-a system, in which hPDGFB and shTP53 transgenes drive tumor initiation. We profiled primary and post-irradiation recurrent tumors, along with healthy brain controls, using the 10x Genomics Flex (GEM-X) gene expression platform and custom probe addon sets targeting the RCAS transcripts. A total of 16 samples were included after QC filtering (primary tumors, recurrent tumors, and control brains). The data set provides genome-wide single-cell RNA-seq measurements at probe-level resolution.2026-03-30T00:00:00Z2026-03-30T01:03:32.563Z2026-01-28T16:41:58.34ZE-MTAB-16532ERP188272EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184