GSM8031699: h_2; Canis lupus familiaris; RNA-Seq
Biopsy samples were rinsed with ice cold phosphate buffered saline (PBS), then centrifuged at 400 rcf for 5 minutes. To dissociate epithelial cells, the samples were digested in Hanks' balanced salt solution (HBSS) with 2 mM ethylenediaminetetraacetic acid (EDTA) and 10% fetal bovine for 30 minutes at 37C with intermittent vortexing. Following the EDTA digestion, the supernatant was removed, passed through a 70-uM cell strainer, and stored in HBSS with 5% FBS and 10 mM 4-(2-Hydroxyethyl)-1-piperazineëthanesulfonic acid (HEPES) on ice. The remaining tissue was resuspended in HBSS with collagenase type II at 250 U/mL and incubated for 30 minutes at 37C with intermittent vortexing. The two cell fractions were pooled together then passed through a 40-uM cell strainer. The pooled cells were resuspended in 4 mls HBSS and layered on top of 3 mls of Ficoll-Paque PLUS. Cells were isolated using density gradient centrifugation to enrich live cells for 30 minutes at 400 rcf with maximum acceleration and no brake. The cellular interface was aspirated then washed with PBS. If cell yield was low, all cells were pooled to obtain adequate cell numbers. To remove contaminating red blood cells, cells were incubated in ammonium-chloride-potassium lysis buffer at room temperature for 3 to 5 minutes. A final 15-minute centrifugation at 100 rcf at 8C was performed to remove small cellular debris and platelets. Lastly, cells were resuspended in 0.04% molecular grade bovine serum in PBS and transported to a Chromium iX instrument for cell capture. All samples were captured within 30 minutes of dissociation. 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 Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.