title: GSM8491769: MS_synovium_12weeks; Mus musculus; RNA-Seq
library_construction_protocol: The articular disk containing synovium was carefully removed from the joint cavity and separated from the surrounding tissue using tweezers and scissors under a stereomicroscope. The synovial membrane was then collected and minced in ice-cold PBS. The minced tissue was subsequently cultured in FCS (fetal calf serum)-free RPMI 1640 medium and digested with constant stirring at 37°C for 15 minutes. The cells were isolated by filtering through 70 μm and 40 μm strainers, followed by centrifugation The library was prepared according to the manufacturer's instructions (single cell 3' v1 protocol, 10x Genomics). Briefly, synovium tissue cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in each individual GEM was reverse transcribed into cDNA, which included 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 the appropriate-sized fragments were selected using SPRIselect reagent for subsequent library construction. An Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added.