{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Lea Wölbert"],"organism":["Mus musculus castaneus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15073"],"description":["The aim of this study was to analyse the evolutionary conservation or divergence of the aging murine hematopoietic system, which includes hematopoietic stem and progenitor cells (HSPCs) and stromal cells from the hematopoietic niche. The animals used were young (10-13 weeks) and old (84-109 weeks) female individuals from four mouse species: Mus musculus strains C57BL/6J or C57BL/6J-Ly5.1 (BL6), Mus musculus castaneus (CAST/EiJ), Mus spretus (SPRET/EiJ), and Mus caroli (CAROLI/EiJ). We isolated HSPC and niche cells from each animal and performed scRNAseq using the 10X Genomics platform. For alignment, we generated a reference genome based on the mm10 genome masked with Ns in positions of SNVs between species."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - scRNAseq was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide, Rev D). 10,000 cells from each individual were loaded separately per channel of the Chromium Single Cell G chip (10X Genomics, Chromium Next GEM Single Cell 3' Reagents Kits v3.1). For some individuals, technical replicates were generated by loading 10,000 niche cells per channel on two or more channels of the chip. Replicates were then processed as separate samples from one individual. Assessment of quality and quantity of cDNA was performed using the TapeStation (Agilent Technologies) and Qubit 4 Fluorometer (Invitrogen) systems.","Nucleic Acid Extraction - scRNAseq was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide, Rev D). 10,000 cells from each individual were loaded separately per channel of the Chromium Single Cell G chip (10X Genomics, Chromium Next GEM Single Cell 3' Reagents Kits v3.1). For some individuals, technical replicates were generated by loading 10,000 niche cells per channel on two or more channels of the chip. Replicates were then processed as separate samples from one individual. Assessment of quality and quantity of cDNA was performed using the TapeStation (Agilent Technologies) and Qubit 4 Fluorometer (Invitrogen) systems.","Sequencing - Libraries were sequenced on Illumina NovaSeq 6000 using paired-end run sequencing 26bp on read 1 and 98 bp on read 2. Each sample had four index variations, and index variations of a sample were then loaded on up to four lanes, each.","Sample Collection - All mice used in this study were derived from colonies maintained in-house at DKFZ. For hematopoietic cell collection, femora were dissected and flushed with FACS buffer (2% FCS (Gibco, A5256801) in PBS (Sigma, D8537)) using a 1ml syringe fitted with a 23 gauge needle. The cell suspension was filtered through a 40µM strainer (Greiner Bio-One, 542040) and treated with Fc blocking agent (anti-CD16/32) (BD Bioscience, 553142, Clone 2.4G2) at 4°C for 30 minutes to reduce unspecific binding, then incubated with primary and secondary antibodies for 30 minutes at 4°C. Cells were then incubated in 1ml ACK lysis buffer (Lonza, 10-548E) at room temperature for 2 minutes for red blood cell lysis. Cells were stained with 7-AAD (Invitrogen, A1310) and up to 24,000 cKit+ or CD45+/cKi","Sample Collection - All mice used in this study were derived from colonies maintained in-house at DKFZ. For stromal cell collection, tibiae, iliac crests, vertebrae, and previously flushed femora were cut into pieces and incubated with digestion buffer (2 mg/ml Collagenase IV (Gibco, 17104019) and 1mg/ml Dispase II (Gibco, 17105041) in HBBS (Gibco, 14175129)) at 37°C for 10 minutes with intermittent shaking. Bone lysate was filtered through a 100µm strainer (Greiner Bio-One, 542000) and the digestion was quenched with FACS buffer. This was repeated a total of three times. Cells were incubated in 1ml ACK lysis buffer at room temperature for either 8 or 10 minutes for red blood cell lysis. Cells were then incubated with FcR blocking agent (anti-CD16/32) at 4°C for 30 minutes to reduce unspec"],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - For alignment of all species, a common reference genome was generated based on the GRCm38 mm10 genome. SNVs between BL6 and non-BL6 species were masked with Ns. SNV positions were derived from Keane et al. 2011 (https://ftp.sanger.ac.uk//pub/mouse_genomes/) and Stefflova et al. 2013 (https://ftp.ebi.ac.uk/pub/databases/vertebrategenomics/FOG09/snp/). All Fastq files from multiple index variations and sequencing lanes per sample were merged and aligned, and gene expression matrices were generated using the Cell Ranger v6.1.1 cellranger count function with default settings. No normalization was performed on processed files.","Data Transformation - For alignment of all species, a common reference genome was generated based on the GRCm38 mm10 genome. SNVs of non-BL6 species were masked with Ns. SNV positions were derived from Keane et al. 2011 (https://ftp.sanger.ac.uk//pub/mouse_genomes/) and Stefflova et al. 2013 (https://ftp.ebi.ac.uk/pub/databases/vertebrategenomics/FOG09/snp/). All Fastq files from multiple index variations and sequencing lanes per sample were merged and aligned, and gene expression matrices were generated using the Cell Ranger v6.1.1 cellranger count function with default settings. No normalization was performed on processed files."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Mus musculus castaneus"],"pubmed_authors":["Lea Wölbert","Veronica Busa","Raphael Coimbra"],"additional_accession":[]},"is_claimable":false,"name":"10X Chromium single-cell RNA sequencing of the hematopoietic system and niche cells of young and old female individuals from four distinct mouse species","description":"The aim of this study was to analyse the evolutionary conservation or divergence of the aging murine hematopoietic system, which includes hematopoietic stem and progenitor cells (HSPCs) and stromal cells from the hematopoietic niche. The animals used were young (10-13 weeks) and old (84-109 weeks) female individuals from four mouse species: Mus musculus strains C57BL/6J or C57BL/6J-Ly5.1 (BL6), Mus musculus castaneus (CAST/EiJ), Mus spretus (SPRET/EiJ), and Mus caroli (CAROLI/EiJ). We isolated HSPC and niche cells from each animal and performed scRNAseq using the 10X Genomics platform. For alignment, we generated a reference genome based on the mm10 genome masked with Ns in positions of SNVs between species.","dates":{"release":"2026-08-15T00:00:00Z","modification":"2026-08-15T15:52:06.291Z","creation":"2025-04-28T11:14:19.59Z"},"accession":"E-MTAB-15073","cross_references":{"ENA":["ERP172022"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}