GSM9571710: PBMCs, untreated; Ovis aries; RNA-Seq
PBMCs were isolated from sheep peripheral blood using HISTOPAQUE 1083 density-gradient separation. The PBMC layer at the interface was collected and washed. Cell number and viability were assessed using a SeekMate Tinitan Fluorescence Cell Counter (SeekGene, M002C) with AO/PI reagent. Debris and dead cells were removed when necessary (Miltenyi, 130-109-398/130-090-101). Cells were resuspended at 1 × 10^6 cells/mL in RPMI 1640 supplemented with 2% FBS for downstream single-cell library preparation. Single-cell RNA-seq libraries were prepared using the SeekOne DD Single Cell 3' library preparation kit (SeekGene, K00202) on a SeekOne chip S3 according to the manufacturer's instructions. Briefly, cells were loaded with reverse transcription reagents, and barcoded hydrogel beads and partitioning oil were used to generate droplets. Reverse transcription was performed to generate barcoded cDNA, followed by droplet breakage, cDNA purification and PCR amplification. Amplified cDNA was then fragmented, end-repaired, A-tailed and ligated to sequencing adapters, and indexed PCR was performed to generate 3' libraries containing cell barcodes and unique molecular identifiers. Libraries were purified with VAHTS DNA Clean Beads (Vazyme, N411-01) and evaluated using Qubit (Thermo Fisher Scientific, Q33226) and a Bio-Fragment Analyzer (Bioptic, Qsep400).