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Sample GSM7779116 Query DataSets for GSM7779116
Status Public on Jan 12, 2024
Title Ldlr-/-Trem2-/-#3
Sample type SRA
Source name Aorta
Organism Mus musculus
Characteristics tissue: Aorta
cell type: nuclei; various lineages
genotype: Ldlr-/-Trem2-/-
Extracted molecule polyA RNA
Extraction protocol Aortas from Ldlr-/- and Ldlr-/-Trem2-/- mice fed a high fat diet for 10 weeks were snap frozen in liquid nitrogen and cryoconserved at -80°C. Nuclei from n=3 mice per group were isolated using Chromium Nuclei Isolation Kits (10x Genomics) according to the manufacturer’s instruction. For counting, nuclei were labelled with DAPI and counted using a fluorescence microscope and a Neubauer counting chamber (concentration range: 1,400-2,300 nuclei/µl). Nuclei from each sample were loaded onto separate lanes of the 10x Genomics Chromium with the aim to recover 10,000 nuclei per sample, using loading volumes recommended by the manufacturer. We employed Chromium Next GEM Single Cell 3’ Kit v3.1 (10x Genomics). Libraries were generated according to the manufacturer’s instructions. All libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). Sequencing was performed using a S1 flowcell with Novaseq 6000 platform (Illumina) targeting 26,500 reads per nucleus.
Single nuclei were encapsulated into droplets with the Chromium™ Controller (10x Genomics). Transcripts captured in all cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 12 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3’ Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer.
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description 3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript
Data processing Sequencing data was demultiplexed and mapped with Cell Ranger software v7.0.1 (10x Genomics). Mouse mm10 (Ensembl 98) reference was used for the alignment and counting steps with intronic reads included
Cell Ranger outputs (filtered_feature_bc_matrix) were loaded in R (version 4.3.1), pre-processed and analyzed using Seurat v4.3.0.1 and DoubletFinder v2.0.3. Each sample was pre-filtered as follows: nuclei with >3% mitochondrial transcripts were excluded, and clustering analysis was performed in Seurat using 20 principal components and a 0.2 resolution. Doublets were excluded using DoubletFinder v2.0.3, inputting a predicted doublet rate of 15%. The six resulting Seurat objects (one for each sample) were pooled. As all the samples were processed simultaneously (same 10x Genomics Chromium chip, sequencing in the same flow cell) and had similar quality control characteristics (number of nuclei recovered, reads per nuclei, UMI per nuclei etc…), the data presented in the manuscript were pooled in Seurat without applying batch correction (replicating the analysis using the batch correction tool Harmony yielded consistent results, not shown). Clustering analysis of all aortic nuclei was performed using 20 principal components and a 0.1 resolution to identify vascular cell lineages. Cells corresponding to mononuclear phagocytes (monocytes, macrophages, dendritic cells) were identified and separately reclustered using 20 principal components and a 0.3 clustering resolution. Cluster markers were identified using ‘FindAllMarkers’ in Seurat.
Assembly: Mouse mm10 (Ensembl 98)
Supplementary files format and content: 10x Genomics output files: barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz
Submission date Sep 13, 2023
Last update date Jan 12, 2024
Contact name Alexander M. Leipold
Organization name Helmholtz Institute for RNA-based Infection Research
Lab Saliba Lab
Street address Josef-Schneider-Str. 2
City Würzburg
ZIP/Postal code 97080
Country Germany
Platform ID GPL24247
Series (1)
GSE243086 Single-nucleus RNA-seq of aortas from Ldlr-/- and Ldlr-/-Trem2-/- atherosclerotic mice
BioSample SAMN37386963
SRA SRX21773427

Supplementary file Size Download File type/resource
GSM7779116_C4_filtered_feature_bc_matrix.tar.gz 85.9 Mb (ftp)(http) TAR
GSM7779116_C4_molecule_info.h5 381.2 Mb (ftp)(http) H5
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