GSM9571458: Testis, Low sperm motility,2; Anser cygnoides; RNA-Seq
Preserved testicular tissue was transferred to a culture dish and washed with precooled 1 × PBS (Biosharp, Beijing, China) to remove residual blood and lipids. The tissue was cut into 0.5 mm² pieces and incubated in a dissociation solution containing 0.35% collagenase IV, 2 mg/mL papain, and 120 U/mL DNase I for 20 min in a 37°C water bath shaker (100 rpm). Digestion was terminated by the addition of 1 × PBS containing 10% fetal bovine serum (Thermo Fisher Scientific, MA, USA). The cell suspension was filtered through a 40-μm cell strainer and centrifuged at 4°C (300 × g, 5 min). Following centrifugation, the pellet was resuspended in red blood cell lysis solution (Miltenyi Biotec, North Rhine-Westphalia, Germany) and centrifuged at 4°C (300 × g, 5 min). Dead cells were removed using a Dead Cell Removal Kit (Miltenyi Biotec, North Rhine-Westphalia, Germany) and passed through MS columns (Miltenyi Biotec, North Rhine-Westphalia, Germany) to eliminate dead cells and residual reagents. The collected cells were centrifuged at 4°C (300 × g, 5 min) and resuspended in 1 × PBS supplemented with 0.04% bovine serum albumin (BSA) (Thermo Fisher Scientific, MA, USA), and then washed twice (each at 4°C, 300 × g, 5 min). The final cell suspension was resuspended in 100 μL of 1 × PBS containing 0.04% BSA to generate a single-cell suspension. Cell viability was assessed using the trypan blue exclusion method, with a threshold of > 85%. Cell counting was performed using a Countess II FL Automated Cell Counter (Thermo Fisher Scientific, MA, USA), and the concentration was adjusted to 700–1200 cells/μL. Single-cell suspensions were loaded onto a Chromium chip (10x Genomics, CA, USA) using a Chromium Single Cell 3′ Reagent Kit v3.1 (10x Genomics, CA, USA), targeting the capture of –8,000 cells, according to the manufacturer's protocol. cDNA amplification and library construction were performed following the standard protocol. The libraries were sequenced with 150-bp paired-end reads on a NovaSeq 6000 platform (Illumina, CA, USA).