{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Fanny Coulpier"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13334"],"description":["Mutant mice (Nf1-KO) develop thicker subcutaneous nerves that progress for some of them in tumors. Analyzing and deciphering the cellular and molecular mechanisms of subcutaneous nerves from control and mutant mice of Neurofibromatosis type 1 at 3 months (before the development of the tumors) aims to identify therapeutic targets for this nerve thickening and tumor progression. To achieve this, subcutaneous nerves were harvested, mechanically and enzymatically dissociated to recover living single cells. The scRNAseq technique (10X genomics) was used to explore the molecular signature of each cell."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - The amplified cDNA was used to generate Illumina sequencing libraries that were each sequenced on one flow cell Nextseq500 Illumina.","Sample Collection - 20 nerves per mouse were dissected and HBSS was added (Sigma, ref: H6648-500ML) on ice. After mechanical dissociation, nerves were transfered in a mix of enzymes (0,15% Trypsin (Sigma #T9201), 0,04% Hyaluronidase (Bioconcept #LS005474), 0,3% Collagenase type 2 (Bioconcept #LS004174) and DNAse I (25 μg/mL)) in HBSS, during 2 hours in a water bath. After digestion, nerves were tritured with a 1000 pipette and a solution of 20% FBS in RPMI (Gibco, ref: 42401018) was added to stop digestion. Tubes were centrifuged and supernatant removed.","Nucleic Acid Extraction - Single viable cells were sorted (BD InfluxTM Cell Sorter) using a nuclear labelling DRAQ7TM Far-Red Fluorescent Live-Cell Impermeant DNA Dye (Abcam, ref: ab109202). Cells were collected in 20% FBS-RPMI solution. Cells were centrifuged, the supernatant removed, and myelin depletion was performed by using anti-myelin beads (Miltenyi Biotec, ref: 130-096-733) in a 0,5% of MACS solution (Miltenyi Biotec, ref: 5130-091-376). Cells were collected after the passage through the magnetic separator. Around 25,000 cells were loaded into one channel of the Chromium system using the V3.1 single cell reagent kit (10X Genomics) to generate single-cell GEMs","Library Construction - Following capture and lysis, cDNA was synthesized, then amplified by PCR for 12 cycles as per the manufacturer’s protocol (10X Genomics)."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - One dataset corresponds to one biological replicate from one condition. Datasets were sequencing independently and not multiplexed. Mapping pipeline generated a raw count matrix for each dataset. Each dataset was analyzed individually. We load the raw count matrices using Seurat V3 package 38. Then, following filters were applied: remove cells with a log number of UMI lower than 6 or expressing a number of genes lower than 600. Then, we removed doublet cells using scDblFinder tool and scds in hybrid mode. Then, cells having more than 20 % of UMI related to mitochondrial genes or more than 30 % of UMI related to ribosomal genes were filtered out. UMI count matrix for remaining cells was normalized using LogNormalize method implemented in Seurat V3 package. From the normalized count matrix, we used AddModuleScore to annotated cells for cell type using the following marker sets : Sox10, Plp1, tdTomato, Nkain2, Ank3 and Kcna1 for Schwann cells ;  Clec3b, Gdf10, Dpt, Gpx3, Slit3, Aebp1, Prrx1, Ly6c1, Pcolce2 and Cxcl14 for epineurial fibroblasts (epiFb) ; Lum, Smoc2, Spp1 and Gpc3 for endoneurial fibroblasts (endoFb) ; Cldn1, Tjp1, Itga6, Ildr2, Zbtb7c, Cttnbp2, Adamtsl3, Fxyd6 and Itgb4 for perineurial fibroblasts (periFb) ; Adgre1, Lyz2, Cd68, Pf4, Csf1r and Apoe for macrophages ; Cd209a, Mgl2, Tnip3, Mcemp1, Cfp, Xcr1 and Napsa for dendritic cells ; Cd3g, Cd3d, Cd3e, Cd8a, Cd4, Foxp3, Gata3 and Areg for T lymphocytes ; Nkg7, Klrk1, Klri2, Klrc2, Klrb1c, Ncr1 and Klrb1f for Natural Killer cells (NK cells) ; Cd79a, Ly6d, Cd79b and Cd19b for B lymphocytes ; Tpsb2, Tpsab1, Cpa3 and Kit for mast cells ; S100a8 and S100a9 for neutrophils ; Egfl7, Flt1, Ptprb, Shank3 and Mecom endothelial cells and Des, (Rgs5, Notch3, Gucy1a1), (Chodl, Nppc, Gal and Myf5) for mural cells. Note that two subpopulations of mural cells were identified, corresponding to the two sets of genes in brackets. We generated a PCA using Seurat's RunPCA function with 100 principal components (PC) from 3,000 highly variable features, identified using Seurat’s FindVariableFeatures function with default settings. From 20 PC, we generated a graph using Seurat’s FindNeighbors function. Finally, we clustered cells from this graph using Seurat’s FindClusters function with a resolution of 2. Individual datasets were then saved for further analyses.","Sequence Alignment - Cell Ranger v7.0.1 (10X Genomics) was used to process raw sequencing data. This pipeline converted Illumina base call files to Fastq format, aligned sequencing reads to a custom mm10 transcriptome with added sequences for the Tomato and Cre transgenes using the STAR aligner (27) and quantified the expression of transcripts in each cell using Chromium barcodes."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["NextSeq 500"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Mus musculus"],"pubmed_authors":["Fanny Coulpier"],"additional_accession":[]},"is_claimable":false,"name":"Single cell RNA sequencing of subcutaneous nerves from 3-month-old control and mutant mice in the context of Neurofibromatosis type 1","description":"Mutant mice (Nf1-KO) develop thicker subcutaneous nerves that progress for some of them in tumors. Analyzing and deciphering the cellular and molecular mechanisms of subcutaneous nerves from control and mutant mice of Neurofibromatosis type 1 at 3 months (before the development of the tumors) aims to identify therapeutic targets for this nerve thickening and tumor progression. To achieve this, subcutaneous nerves were harvested, mechanically and enzymatically dissociated to recover living single cells. The scRNAseq technique (10X genomics) was used to explore the molecular signature of each cell.","dates":{"release":"2025-08-30T00:00:00Z","modification":"2025-08-30T01:01:54.186Z","creation":"2023-09-07T13:35:25.764Z"},"accession":"E-MTAB-13334","cross_references":{"ENA":["ERP151015"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}