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Sample GSM6742839 Query DataSets for GSM6742839
Status Public on Mar 01, 2023
Title Dog2_abTcells_VDJ
Sample type SRA
 
Source name peripheral T cells, TCRab+
Organism Canis lupus familiaris
Characteristics breed: Beagle
name: Theo
Sex: male
age: 7
cell type: peripheral T cells, TCRab+
genotype: healthy
treatment: NA
Extracted molecule polyA RNA
Extraction protocol Venous blood was taken from 4 healthy experimental Beagle dogs (one female, three male, age: 5 – 9 years) into heparinized vacutainer tubes (The study was authorized by the Animal Care and Usage Committee of the Saxony State Office (Landesdirektion Sachsen) in Leipzig, Germany; approval number: A 28/18). PBMC were isolated by density gradient centrifugation and stained with the fixable viability dye eFluor 780 (Thermo Fisher Scientific, Carlsbad, USA) according to the manufacturer’s protocol to discriminate dead from viable cells. In a second step, they were incubated with a mixture of heat-inactivated normal serum derived from dog and rat (each 15% in PBS) to block unspecific binding of Fc receptors. Then, cells were incubated with anti-canine TCRαβ (clone CA15.8G7) hybridoma supernatant, for 15 min in the dark on ice. A PerCP/Cy5.5-conjugated goat-anti-mouse IgG secondary antibody (Biolegend, San Diego, USA) was used for detection. After exclusion of dead cells and doublets, TCRαβ+ T cells from the lymphocyte gate were sorted using the BD FACSAriaTM III flow cytometer (Becton Dickinson, Heidelberg, Germany) with a purity >99% (Re-analysis using the FlowJoTM10 software (Treestar Inc., Ashland, OR, USA). Isolated TCRαβ+ T cells were cryopreserved (FCS, 10% DMSO) and shipped to the Institute of Pathology, Microbiology & Immunology of the UC Davis. There, the cellularity (1.1-1.4 x 106 cells/ml) and viability (78-90%) of TCRαβ+ T cells was confirmed.
Transcriptome and V(D)J libraries were prepared at the UC Davis Genome Center. Briefly, ~10,000 TCRαβ+ T cells per lane were loaded on a Chromium Single Cell Controller (10× Genomics). Captured cells were lysed, and the released RNAs were barcoded through reverse transcription in individual gel beads in emulsion (GEMs). Starting mRNA molecules were indexed using Unique Molecular Identifier (UMI). Following emulsion breakage, barcoded cDNA was amplified and libraries were constructed using the Chromium Next GEM Single Cell 5' Kit v2. The canine T cell receptor alpha (TRA) and T cell receptor beta (TRB) genes were amplified using the Chromium Single Cell V(D)J Enrichment Kit according to the manufacturer’s instructions but with primers adapted for the canine genes. Libraries were sequenced using the Illumina NovaSeq S4 platform using 150 paired-end reads to a target read depth of 30,000 reads per cell.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description PE150
Data processing The demultiplexing, barcoded processing, gene counting and aggregation were performed using the Cell Ranger software version cellranger 5.0.1
Assembly: CanFam 3.1 annotation genebuild updated 2019-06, GenBank assembly accession: GCA_000002285
Supplementary files format and content: Raw data: 1) Transcriptome: bam files; 2) VDJ: bam files
Supplementary files format and content: Processed data: 1) Transcriptome: CellRanger output files (barcodes, features, matrix files); 2) VDJ: CellRanger output files (airr format)
 
Submission date Nov 18, 2022
Last update date Mar 03, 2023
Contact name Stefan Matthias Keller
E-mail(s) smkeller@ucdavis.edu
Organization name University of California, Davis
Department Pathology, Immunology, Microbiology
Street address One Shields Avenue
City Davis
State/province California
ZIP/Postal code 95616
Country USA
 
Platform ID GPL25760
Series (1)
GSE218355 Canine peripheral blood TCRαβ T cell atlas: Identification of diverse subsets including CD8+ MAIT-like cells by combined single-cell transcriptome and V(D)J repertoire analysis
Relations
BioSample SAMN31792052
SRA SRX18312921

Supplementary file Size Download File type/resource
GSM6742839_VDJ_DOG2_Theo.tsv.gz 2.5 Mb (ftp)(http) TSV
GSM6742839_all_contig_DOG2_Theo.fasta.gz 1.6 Mb (ftp)(http) FASTA
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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