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Sample GSM6372185

Query DataSets for GSM6372185

Status

Public on Jul 22, 2022

Title

C57BL/6J Blood Old, GEX

Sample type

SRA

Source name

blood

Organism

Mus musculus

Characteristics

strain: C57BL/6J
developmental stage: Old
age: 88 weeks
tissue: blood
cell type: PBMC
cell subset: CD8+ T cells
genotype: WT

Growth protocol

Mice were bred in SPF Facility (Institute of Molecular Genetics of the Czech Academy of Sciences, IMG) in accordance with the laws of the Czech Republic and were kept in the animal facility with 12 hours of light and dark cycle with food and water ad libitum. A group of young (n = 3, age = 8 weeks) and old (n = 3, age = 88 week) was used for this experiment.

Extracted molecule

total RNA

Extraction protocol

Mice were anesthetized using an i.p. injection of Ketamine-Xylazine. Carotid artery blood was collected to individual EDTA-coated tubes supplemented with 100 μL of 3.5mM EDTA, blood from each mice age group was pooled, spinned down (4 °C, 400 g, 5 min) and the supernatant was removed. Cells were lysed in 5 mL of ACK buffer for 3 minutes in room temperature, and the reaction was stopped by adding 10mL of PBS. The lysis step was repeated once more. Cells were stained on ice in darkness in 200 µL of PBS supplemented with 2% FBS and 400× diluted feature barcoding antibody markers to distinguish cells from this experiment from another, unrelated one. After 10 min of staining, 200× diluted mouse anti-CD5 and anti-CD8α were added for additional 20 min. CD8α+ CD5+ cells were sorted on an Influx.
The gene expression and cell surface protein libraries were constructed following Feature Barcode technology for Cell Surface Protein protocol (#CG000186 Rev D) with the Chromium Single Cell 5’ Library & Gel Bead and Chromium Single Cell 5' Feature Barcode Library kits (10x Genomics, #PN-1000014, #PN-1000020, #PN-1000080, #PN-1000009, #PN-1000084). The V(D)J library was built following the same protocol with Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell kit (10X Genomics, #PN-1000071).
scRNAseq (10x Genomics Single Cell Immune Profiling with Feature Barcoding)

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

b6_blood_integrated.rds

Data processing

The initial demultiplexing, barcode processing, GEX counting, V(D)J counting and cell surface marker barcode identification was performed using Cell Ranger v5.0.0. (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/5.0/what-is-cell-ranger).
The data were demultiplexed using cell surface protein marker counts, on which an in-house R script was applied to determine cells (and cellular barcodes corrected by Cellranger) belonging to this experiment and separate them from another, unrelated one.
The mapping and counting of separated experiment was performed using Cell Ranger v5.0.0.
Analysis, integration of data from young and old mice, integration of V(D)J data, count normalization, variable feature detection, dimensional reduction and clustering was performed using Seurat package v4.0.3 on R v4.0.4.
Assembly: GRCm38 (Ensembl release 102)
Supplementary files format and content: rds file of analyzed data-set obtained by integration of all samples, in form of SeuratObject containing log-normalized counts, detected variable features, UMAP and PCA dimensional reductions, clustering, mice-relevant annotation and V(D)J information.

Submission date

Jul 21, 2022

Last update date

Jul 22, 2022

Contact name

Juraj Michalik

E-mail(s)

juraj.michalik@img.cas.cz

Organization name

Institute of Molecular Genetics of the Czech Academy of Sciences

Lab

Lab of Adaptive Immunity