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Status |
Public on Nov 01, 2020 |
Title |
56 cns_wt_cd4 |
Sample type |
SRA |
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|
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).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
NextSeq 550 |
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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
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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 |