GSM8058731: syn_N; Equus caballus; RNA-Seq
Synovial fluid (5 to 10 mL) was sampled from the affected middle carpal joint (osteoarthritic sample) and tibiotarsal joint (normal sample). Synoviocenteses to obtain synovial fluid were performed implementing aseptic technique using sterile gloves, needles (20 gauge, 1.5 inches), and syringes (23). Synovial fluid samples were washed with PBS, filtered through a 40 µM cell strainer, then red blood cells were removed using Ammonium-Chloride-Potassium (ACK) lysis buffer. Red blood cell depleted suspensions were subsequently resuspended in PBS with 0.04% molecular grade BSA (Sigma-Aldrich; St. Louis, MO).  Synovium was obtained immediately following euthanasia.  Briefly, the horse was euthanized with pentobarbital (1 mL / 5 kg body weight) administered via indwelling catheter, then the middle carpal and tibiotarsal joints were dissected aseptically and synovium was sampled from four quadrants of each joint (dorsomedial, dorsolateral, palmar- or plantaromedial, palmar- or plantarolateral) and pooled for analysis. Synovial tissue samples were then disaggregated into single cell suspensions. Briefly, synovial tissue fragments were separated mechanically and enzymatically into digestion buffer (Liberase TL (Sigma-Aldrich) 100 µg/mL and DNAase I (New England Biolabs) 100 µg/mL in RPMI) in a 37°C water bath for 30 minutes. Digested suspensions were washed with PBS then resuspended in PBS with 0.04% non-acetylated BSA (Sigma-Aldrich). To remove small debris and platelets, a final wash at 100 rcf for 15 minutes was completed. The samples were then prepared to be run on a Chomium iX instument by resusspending 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 Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.