GSM7050918: OS4; Canis lupus familiaris; RNA-Seq
Approximately 10-mL of canine whole blood was obtained in EDTA collection tubes then processed within 30 minutes using Ficoll Paque to complete density gradient centrifugation. Whole blood was mixed with 12 mL of phosphate buffered saline, pH = 7.40, then layered onto 12 mL of Ficoll Paque. To isolated leukocytes, the layered sample was then centrifugated at room temperature for 40 minutes at 500 rcf with acceleration at maximum and brake off. Cells were washed, red blood cells lysed, and the sample prepared to be run on a Chromium iX instrument by resuspending in 0.04% molecular grade BSA. The instrument was loaded to target 5,000 cells per sample. Library construction was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3ʹ Kit v3.1 protocol, 10x Genomics). Briefly, a Chromium iX instrument was used to pass cells and oligonucleotide coated gel beads at a limiting dilution to obtain a coupling of cells and beads in a 1:1 ratio. The cell-bead pair was then passed from an aqueous phase to an oil phase to form an emulsion. Within the emulsions, cells were lysed, and the oligonucleotide sequences on the gel beads pull down message RNA and other RNA molecules that have long adenosine stretches with an oligo-d(T) pull down. After hybridization, reverse transcription is completed to generate a cDNA library with each transcript now associated with a unique cell barcode and an Illumina R1 primer sequence.  Once cells were barcoded and unique molecular identifiers (UMIs) added, a standard Illumina library preparation kit was completed with SPRIselect used for fragment size selection. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.