title: GSM9659754: aged mouse synovial cells; Mus musculus; RNA-Seq
library_name: GSM9659754
library_construction_protocol: Primary synovial cells were isolated from the knee joints of 24‑month‑old wild‑type (WT) mice. The synovial tissues were carefully dissected, minced, and sequentially digested with 0.25% trypsin (Thermo Fisher, 25200072, USA) for 30 min, followed by type I collagenase (1 mg/mL, Thermo Fisher, 17100017, USA) for 45 min at 37 °C with constant agitation. The digestion was stopped by adding complete culture medium. The resulting cell suspension was filtered, centrifuged, and the pellet was resuspended in DMEM supplemented with 10% heat‑inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) for further use. Library preparation was performed according to the manufacturer's instructions (Single Cell 3′ v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate gel beads-in-emulsion (GEMs). The poly-A RNA from the cell lysate contained in each single GEM was reverse transcribed into cDNA, which incorporates an Illumina R1 primer sequence, a unique molecular identifier (UMI), and the 10x barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR, and appropriately sized fragments were selected using SPRIselect reagent for subsequent library construction. During library construction, the Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added.
identifiers.namespace: GEO
identifiers.id: GSM9659754_r1