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Sample GSM4826916 Query DataSets for GSM4826916
Status Public on Nov 01, 2020
Title 56 cns_wt_cd4
Sample type SRA
 
Source name CD4 T cells
Organism Mus musculus
Characteristics background strain: B6
treatment: MOG35-55 peptide
genotype/variation: Lag3wt
Treatment protocol CD45+ or CD4+ lymphocytes were enriched by positive bead selection (Miltenyi Biotec).
Growth protocol EAE was induced in aged and sex matched control (Lag3WT) or LAG3 deficient (Lag3Flox) mice groups (5 mice per group). At the peak of disease, mice with matching disease scores in each group were sacrificed and CNS cells isolated
Extracted molecule total RNA
Extraction protocol Single cell suspensions were resuspended in RPMI plus 2% FBS at a concentration of 1x106 cells/mL and barcoded with a 10x Chromium Controller (10x Genomics) according to the manufacturer’s instructions. For each sample, we loaded 8.7µl with goal recovery of 5000 cells.
RNA from the barcoded cells for each sample was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a Chromium Single Cell 3′ v2 reagent kit (10x Genomics) according to the manufacturer’s instructions. Libraries were quantified and assessed for quality by Tape Station. Sequencing was performed with Illumina NextSeq according to the manufacturer’s instructions (Illumina).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model NextSeq 550
 
Data processing The Cell Ranger analysis pipeline v3.1 was used for sample demultiplexing, barcode processing, alignment, filtering, unique molecular identifier counting and aggregation of sequencing runs
Downstream analysis such as shared nearest neighbor graph-based clustering, differential expression analysis and visualization—were performed using the R package Seurat
A filtered gene barcode matrix of all samples was integrated with Seurat. Normalization of the gene barcode matrix was done using LogNormalize method with default parameters, the FindVariableFeatures function in Seurat was used to identify top 2000 variable genes, PCA was performed using the top variable genes and tSNE and UMAP was performed on the top principal components for visualizing the cells in two dimensional space,Graph-based clustering was performed for clustering analysis in Seurat
Differential expression analysis of scRNAseq data was performed using wilcox rank sum test in Seurat (FindMarkers function) to identify gene markers for a cluster/group. A gene was considered significant with adjusted P<0.05
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: Filtered gene-barcode matrices containing only cellular barcodes
 
Submission date Oct 09, 2020
Last update date Nov 02, 2020
Contact name E JOHN WHERRY
Organization name University of Pennsylvania
Department Systems Pharmacology and Translational Therapeutics,Institute for Immunology
Street address 421 Curie Blvd,354 BRB II/III
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
Platform ID GPL21626
Series (1)
GSE159342 Transcriptional analysis of LAG3 deficient Tregs in an inflamed CNS
Relations
BioSample SAMN16408690
SRA SRX9273096

Supplementary file Size Download File type/resource
GSM4826916_56_barcodes.tsv.gz 9.0 Kb (ftp)(http) TSV
GSM4826916_56_features.tsv.gz 221.0 Kb (ftp)(http) TSV
GSM4826916_56_matrix.mtx.gz 6.8 Mb (ftp)(http) MTX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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