GSM6742841: Dog3_abTcells_VDJ; Canis lupus familiaris; RNA-Seq
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.