title: GSM8001771: N_4; Canis lupus familiaris; RNA-Seq
library_construction_protocol: Between 3 and 5 tumor biopsies were collected from the amputated limb, washed with phosphate buffered saline (PBS), minced using a scalpel, and digested with collagenase type II (250 U/mL) in Hanks' Balanced Salt Solution (HBSS) for 45 minutes at 37˚C with agitation (Thermo Fisher Scientific Inc.). Samples were passed through a 70-µm cell strainer, washed with PBS, then centrifuged for 5 minutes at 400 rcf. To enrich for live cells, samples were pooled into 4-mL HBSS layered onto 3-mL Ficoll Paque (Cytiva; Marlborough, MA), and centrifuged for 30 minutes at 400 rcf with acceleration at 9 and brake at 0. Following density centrifugation, the cell interface layer was collected, washed one time with PBS, and resuspended in 10-mL of Ammonium-Chloride-Potassium lysis buffer for 3-7 minutes at room temperature. To remove small debris and platelets, a final wash at 100 rcf for 15 minutes was completed. The sample was then prepared to be run on a Chomium iX instument by resusspending in 0.04% molecular grade BSA. The instumnet 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 Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.