title: GSM9086671: Healthy 1 canine PBMC single-cell landscape; Canis lupus familiaris; RNA-Seq
library_construction_protocol: Blood was collected via jugular venipuncture and processed immediately. Peripheral leukocytes or peripheral blood mononuclear cells were isolated by density gradient centrifugation using SepMate 15 tubes (Stemcell Technologies, Vancouver, Canada) and Ficoll Paque PLUS (GE Healthcare, Chicago, IL, USA). The PBMC fraction was washed twice with Dulbecco's phosphate buffered saline (DPBS; Thermo Fisher Scientific, Waltham, MA, USA) containing 2% heat inactivated fetal bovine serum (FBS; Life Technologies, Pleasanton, CA, USA). A small aliquot of the suspension was cytospun onto glass slides and stained with Diff Quik to confirm cell composition. The remaining PBMCs were cryopreserved in a cytoprotective medium—either CryoStor CS10 (Stemcell Technologies) or Cellbanker 1 (Zenogen Pharma, Koriyama, Japan), and stored in liquid nitrogen until further analysis. Thawed PBMCs were washed and incubated on ice for 10 minutes with an anti dog Fc receptor blocking reagent (Invitrogen, eBioscience, San Diego, CA, USA). After washing with DPBS containing 2% FBS, cells were stained for 30 minutes at 37 °C, protected from light, with Fixable Viability Dye eFluor 780 (Invitrogen, eBioscience), anti dog CD45 FITC (clone YKIX 716.13; Bio Rad Laboratories, Hercules, CA, USA), and anti dog CD3 FITC (clone CA17.2A12; Bio Rad Laboratories). Live single cells were then sorted on a FACSAria III (BD Biosciences Pharmingen, San Diego, CA, USA), gating on CD45⁺CD3⁺ T cells and CD45⁺CD3⁻ non T leukocytes. Post sort viability and cell counts were assessed using a LUNA FL™ Automated Fluorescence Cell Counter (Logos Biosystems, Anyang, South Korea). Equal numbers of CD45⁺CD3⁺ and CD45⁺CD3⁻ cells from each dog were pooled and immediately processed for single cell library construction on the 10x Genomics Chromium platform in a single run. Single cell libraries were prepared using the Chromium Next GEM Single Cell 5′ Kit v2 (10x Genomics) following the manufacturer's protocol 38,39. Libraries were sequenced on an Illumina NovaSeq 6000 with 2 × 150 bp paired end reads.